Tetrodotoxin: A New Strategy to Treat Visceral Pain?

Visceral pain is one of the most common symptoms associated with functional gastrointestinal (GI) disorders. Although the origin of these symptoms has not been clearly defined, the implication of both the central and peripheral nervous systems in visceral hypersensitivity is well established. The role of several pathways in visceral nociception has been explored, as well as the influence of specific receptors on afferent neurons, such as voltage-gated sodium channels (VGSCs). VGSCs initiate action potentials and dysfunction of these channels has recently been associated with painful GI conditions. Current treatments for visceral pain generally involve opioid based drugs, which are associated with important side-effects and a loss of effectiveness or tolerance. Hence, efforts have been intensified to find new, more effective and longer-lasting therapies. The implication of VGSCs in visceral hypersensitivity has drawn attention to tetrodotoxin (TTX), a relatively selective sodium channel blocker, as a possible and promising molecule to treat visceral pain and related diseases. As such, here we will review the latest information regarding this toxin that is relevant to the treatment of visceral pain and the possible advantages that it may offer relative to other treatments, alone or in combination.

From Poison to Promise: The Evolution of Tetrodotoxin and Its Potential as a Therapeutic.

Tetrodotoxin (TTX) is a potent neurotoxin that was first identified in pufferfish but has since been isolated from an array of taxa that host TTX-producing bacteria. However, determining its origin, ecosystem roles, and biomedical applications has challenged researchers for decades. Recognized as a poison and for its lethal effects on humans when ingested, TTX is primarily a powerful sodium channel inhibitor that targets voltage-gated sodium channels, including six of the nine mammalian isoforms. Although lethal doses for humans range from 1.5-2.0 mg TTX (blood level 9 ng/mL), when it is administered at levels far below LD50, TTX exhibits therapeutic properties, especially to treat cancer-related pain, neuropathic pain, and visceral pain. Furthermore, TTX can potentially treat a variety of medical ailments, including heroin and cocaine withdrawal symptoms, spinal cord injuries, brain trauma, and some kinds of tumors. Here, we (i) describe the perplexing evolution and ecology of tetrodotoxin, (ii) review its mechanisms and modes of action, and (iii) offer an overview of the numerous ways it may be applied as a therapeutic. There is much to be explored in these three areas, and we offer ideas for future research that combine evolutionary biology with therapeutics. The TTX system holds great promise as a therapeutic and understanding the origin and chemical ecology of TTX as a poison will only improve its general benefit to humanity.

Tetrodotoxin for Chemotherapy-Induced Neuropathic Pain: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Dose Finding Trial.

Tetrodotoxin (TTX) has emerged as a potentially efficacious agent for chemotherapy-induced neuropathic pain (CINP), a prevalent, debilitating condition often resistant to analgesics. This randomized, double-blind, dose-finding study was undertaken to explore safety and trends in efficacy of four TTX doses and to identify a dose for further study. One hundred and twenty-five patients with taxane- or platinum-related CINP received subcutaneous placebo or TTX (7.5 µg twice daily (BID), 15 µg BID, 30 µg once daily (QD), 30 µg BID) for four consecutive days. Primary outcome measure was average patient-reported Numeric Pain Rating Scale (NPRS) score during Days 21-28 post-treatment. Changes in mean NPRS score were not statistically different between cohorts, due to small trial size and influence of a few robust placebo responders. Cumulative responder analysis showed significant difference from placebo with 30 µg BID cohort using the maximum response at any timepoint (p = 0.072), 5-day (p = 0.059), 10-day (p = 0.027), and 20-day (p = 0.071) rolling averages. In secondary quality of life (QOL) outcomes, 30 µg BID cohort also differed significantly from placebo in a number of SF-36 and CIPN20 subscales. Most adverse events (AE) were mild or moderate with oral paresthesia (29.6%) and oral hypoesthesia (24.8%) as most common.

Revisited - Failure of tetrodotoxin to protect red-spotted newts, Notophthalmus viridescens, from endoparasites.

Red-spotted newts, Notophthalmus viridescens, contain tetrodotoxin (TTX) and its analogue 6-epiTTX in variable concentrations. In a follow-up study, newts were sampled from a pond in Pennsylvania, USA, in 2010, 2014, and 2018. Their toxin levels were assayed by liquid-chromatography-fluorescence detection (LC-FLD), and assessment of their infection with endoparasites such as nematodes and helminths was performed by histological examination of internal organs. In the 2010 and 2014 samples, average prevalence of parasite infection was 53 and 60%, respectively, but reached 100% in the 2018 sample, where metacercaria stages of the digenean trematode genus Australapatemon/Apatemon (family: Strigeidae) were predominant causing severe tissue damage in liver and kidney. Mean values of TTX and 6-epiTTX were not significantly different in parasitized or parasite-free newts over the study period, confirming previous findings that host toxicity and parasite load are not negatively correlated. Whereas the role of TTX in defence against predators is undisputed, its efficacy to prevent parasitic infections is less obvious. Toxin-resistance of various metazoan parasites may promote their widespread occurrence in poisonous newts.

Addressing the Issue of Tetrodotoxin Targeting.

This review is devoted to the medical application of tetrodotoxin (TTX), a potent non-protein specific blocker of voltage-gated sodium (NaV) channels. The selectivity of action, lack of affinity with the heart muscle NaV channels, and the inability to penetrate the blood⁻brain barrier make this toxin an attractive candidate for anesthetic and analgesic drug design. The efficacy of TTX was shown in neuropathic, acute and inflammatory pain models. The main emphasis of the review is on studies focused on the improvement of TTX efficacy and safety in conjunction with additional substances and drug delivery systems. A significant improvement in the effectiveness of the toxin was demonstrated when used in tandem with vasoconstrictors, local anesthetics and chemical permeation enhancers, with the best results obtained with the encapsulation of TTX in microparticles and liposomes conjugated to gold nanorods.

Acute engagement of Gq-mediated signaling in the bed nucleus of the stria terminalis induces anxiety-like behavior.

The bed nucleus of the stria terminalis (BNST) is a brain region important for regulating anxiety-related behavior in both humans and rodents. Here we used a chemogenetic strategy to investigate how engagement of G protein-coupled receptor (GPCR) signaling cascades in genetically defined GABAergic BNST neurons modulates anxiety-related behavior and downstream circuit function. We saw that stimulation of vesicular γ-aminobutyric acid (GABA) transporter (VGAT)-expressing BNST neurons using hM3Dq, but neither hM4Di nor rM3Ds designer receptors exclusively activated by a designer drug (DREADD), promotes anxiety-like behavior. Further, we identified that activation of hM3Dq receptors in BNST VGAT neurons can induce a long-term depression-like state of glutamatergic synaptic transmission, indicating DREADD-induced changes in synaptic plasticity. Further, we used DREADD-assisted metabolic mapping to profile brain-wide network activity following activation of Gq-mediated signaling in BNST VGAT neurons and saw increased activity within ventral midbrain structures, including the ventral tegmental area and hindbrain structures such as the locus coeruleus and parabrachial nucleus. These results highlight that Gq-mediated signaling in BNST VGAT neurons can drive downstream network activity that correlates with anxiety-like behavior and points to the importance of identifying endogenous GPCRs within genetically defined cell populations. We next used a microfluidics approach to profile the receptorome of single BNST VGAT neurons. This approach yielded multiple Gq-coupled receptors that are associated with anxiety-like behavior and several potential novel candidates for regulation of anxiety-like behavior. From this, we identified that stimulation of the Gq-coupled receptor 5-HT2CR in the BNST is sufficient to elevate anxiety-like behavior in an acoustic startle task. Together, these results provide a novel profile of receptors within genetically defined BNST VGAT neurons that may serve as therapeutic targets for regulating anxiety states and provide a blueprint for examining how G-protein-mediated signaling in a genetically defined cell type can be used to assess behavior and brain-wide circuit function.

Enhanced Triggering of Local Anesthetic Particles by Photosensitization and Photothermal Effect Using a Common Wavelength.

On-demand pain relief systems would be very helpful additions to the armamentarium of pain management. Near-infrared triggered drug delivery systems have demonstrated the potential to provide such care. However, challenges remain in making such systems as stimulus-sensitive as possible, to enhance depth of tissue penetration, repeatability of triggering, and safety. Here we developed liposomes containing the local anesthetic tetrodotoxin and also containing a photosensitizer and gold nanorods that were excitable at the same near-infrared wavelength. The combination of triggering mechanisms enhanced the photosensitivity and repeatability of the system in vitro when compared with liposomes with a single photoresponsive component. In vivo, on-demand local anesthesia could be induced with a low irradiance and short irradiation duration, and liposomes containing both photosensitizer and gold nanorods were more effective than those containing just one photoresponsive component. Tissue reaction was benign.

Tetrodotoxin for Moderate to Severe Cancer-Related Pain: A Multicentre, Randomized, Double-Blind, Placebo-Controlled, Parallel-Design Trial.

OBJECTIVE: This study evaluated subcutaneous injections of tetrodotoxin (TTX) for the treatment of moderate to severe, inadequately controlled cancer-related pain. METHODS: Eligible patients were randomized to receive TTX (30 µg) or placebo subcutaneously twice daily for four consecutive days. Efficacy was assessed using pain and composite endpoints (including pain and quality of life measures), and safety was evaluated using standard measures. RESULTS: 165 patients were enrolled at 19 sites in Canada, Australia, and New Zealand, with 149 patients in the primary analysis "intent-to-treat" population. The primary analysis supports a clinical benefit of TTX over placebo based on the pain endpoint alone with a clinically significant estimated effect size of 16.2% (p = 0.0460). The p value was nominally statistically significant after prespecified (Bonferroni Holm) adjustment for the two primary endpoints but not at the prespecified two-sided 5% level. The mean duration of analgesic response was 56.7 days (TTX) and 9.9 days (placebo). Most common adverse events were nausea, dizziness, and oral numbness or tingling and were generally mild to moderate and transient. CONCLUSIONS: Although underpowered, this study demonstrates a clinically important analgesic signal. TTX may provide clinically meaningful analgesia for patients who have persistent moderate to severe cancer pain despite best analgesic care. This clinical study is registered with ClinicalTrials.gov (NCT00725114).

Tetrodotoxin suppresses thermal hyperalgesia and mechanical allodynia in a rat full thickness thermal injury pain model.

Burn injuries have been identified as the primary cause of injury in 5% of U.S. military personnel evacuated from Operations Iraqi Freedom and Enduring Freedom. Severe burn-associated pain is typically treated with opioids such as fentanyl, morphine, and methadone. Side effects of opioids include respiratory depression, cardiac depression, decrease in motor and cognitive function, as well as the development of hyperalgesia, tolerance and dependence. These effects have led us to search for novel analgesics for the treatment of burn-associated pain in wounded combat service members. Tetrodotoxin (TTX) is a selective voltage-gated sodium channel blocker currently in clinical trials as an analgesic. A phase 3 clinical trial for cancer-related pain has been completed and phase 3 clinical trials on chemotherapy-induced neuropathic pain are planned. It has also been shown in mice to inhibit the development of chemotherapy-induced neuropathic pain. TTX was originally identified as a neurotoxin in marine animals but has now been shown to be safe in humans at therapeutic doses. The antinociceptive effects of TTX are thought to be due to inhibition of Na(+) ion influx required for initiation and conduction of nociceptive impulses. One TTX sensitive sodium channel, Nav1.7, has been shown to be essential in lowering the heat pain threshold after burn injuries. To date, the analgesic effect of TTX has not been tested in burn-associated pain. Male Sprague-Dawley rats were subjected to a full thickness thermal injury on the right hind paw. TTX (8 µg/kg) was administered once a day systemically by subcutaneous injection beginning 3 days post thermal injury and continued through 7 days post thermal injury. Thermal hyperalgesia and mechanical allodynia were assessed 60 and 120 min post injection on each day of TTX treatment. TTX significantly reduced thermal hyperalgesia at all days tested and had a less robust, but statistically significant suppressive effect on mechanical allodynia. These results suggest that systemic TTX may be an effective, rapidly acting analgesic for battlefield burn injuries and has the potential for replacing or reducing the need for opioid analgesics.

The scorpion toxin Amm VIII induces pain hypersensitivity through gain-of-function of TTX-sensitive Na⁺ channels.

Voltage-gated Na(+) channels (Nav) are the targets of a variety of scorpion toxins. Here, we investigated the effects of Amm VIII, a toxin isolated from the venom of the scorpion Androctonus mauretanicus mauretanicus, on pain-related behaviours in mice. The effects of Amm VIII were compared with the classic scorpion α-toxin AaH II from Androctonus australis. Contrary to AaH II, intraplantar injection of Amm VIII at relatively high concentrations caused little nocifensive behaviours. However, Amm VIII induced rapid mechanical and thermal pain hypersensitivities. We evaluated the toxins' effects on Nav currents in nociceptive dorsal root ganglion (DRG) neurons and immortalized DRG neuron-derived F11 cells. Amm VIII and AaH II enhanced tetrodotoxin-sensitive (TTX-S) Nav currents in DRG and F11 cells. Both toxins impaired fast inactivation and negatively shifted activation. AaH II was more potent than Amm VIII at modulating TTX-S Nav currents with EC50 of 5 nM and 1 µM, respectively. AaH II and Amm VIII also impaired fast inactivation of Nav1.7, with EC50 of 6.8 nM and 1.76 µM, respectively. Neither Nav1.8 nor Nav1.9 was affected by the toxins. AaH II and Amm VIII reduced first spike latency and lowered action potential threshold. Amm VIII was less efficient than AaH II in increasing the gain of the firing frequency-stimulation relationship. In conclusion, our data show that Amm VIII, although less potent than AaH II, acts as a gating-modifier peptide reminiscent of classic α-toxins, and suggest that its hyperalgesic effects can be ascribed to gain-of-function of TTX-S Na(+) channels in nociceptors.

Mitofusin2 mutations disrupt axonal mitochondrial positioning and promote axon degeneration.

Alterations in mitochondrial dynamics (fission, fusion, and movement) are implicated in many neurodegenerative diseases, from rare genetic disorders such as Charcot-Marie-Tooth disease, to common conditions including Alzheimer's disease. However, the relationship between altered mitochondrial dynamics and neurodegeneration is incompletely understood. Here we show that disease associated MFN2 proteins suppressed both mitochondrial fusion and transport, and produced classic features of segmental axonal degeneration without cell body death, including neurofilament filled swellings, loss of calcium homeostasis, and accumulation of reactive oxygen species. By contrast, depletion of Opa1 suppressed mitochondrial fusion while sparing transport, and did not induce axonal degeneration. Axon degeneration induced by mutant MFN2 proteins correlated with the disruption of the proper mitochondrial positioning within axons, rather than loss of overall mitochondrial movement, or global mitochondrial dysfunction. We also found that augmenting expression of MFN1 rescued the axonal degeneration caused by MFN2 mutants, suggesting a possible therapeutic strategy for Charcot-Marie-Tooth disease. These experiments provide evidence that the ability of mitochondria to sense energy requirements and localize properly within axons is key to maintaining axonal integrity, and may be a common pathway by which disruptions in axonal transport contribute to neurodegeneration.

Tetrodotoxin (TTX) as a therapeutic agent for pain.

Tetrodotoxin (TTX) is a potent neurotoxin that blocks voltage-gated sodium channels (VGSCs). VGSCs play a critical role in neuronal function under both physiological and pathological conditions. TTX has been extensively used to functionally characterize VGSCs, which can be classified as TTX-sensitive or TTX-resistant channels according to their sensitivity to this toxin. Alterations in the expression and/or function of some specific TTX-sensitive VGSCs have been implicated in a number of chronic pain conditions. The administration of TTX at doses below those that interfere with the generation and conduction of action potentials in normal (non-injured) nerves has been used in humans and experimental animals under different pain conditions. These data indicate a role for TTX as a potential therapeutic agent for pain. This review focuses on the preclinical and clinical evidence supporting a potential analgesic role for TTX. In addition, the contribution of specific TTX-sensitive VGSCs to pain is reviewed.

Rapid synaptic remodeling in the adult somatosensory cortex following peripheral nerve injury and its association with neuropathic pain.

Structural and functional plastic changes in the primary somatosensory cortex (S1) have been observed following peripheral nerve injury that often leads to neuropathic pain, which is characterized by tactile allodynia. However, remodeling of cortical connections following injury has been believed to take months or years; this is not temporally correlated with the rapid development of allodynia and S1 hyperexcitability. Here we first report, by using long-term two-photon imaging of postsynaptic dendritic spines in living adult mice, that synaptic connections in the S1 are rewired within days following sciatic nerve ligation through phase-specific and size-dependent spine survival/growth. Spine turnover in the S1 area corresponding to the injured paw markedly increased during an early phase of neuropathic pain and was restored in a late phase of neuropathic pain, which was prevented by immediate local blockade of the injured nerve throughout the early phase. New spines that generated before nerve injury showed volume decrease after injury, whereas more new spines that formed in the early phase of neuropathic pain became persistent and substantially increased their volume during the late phase. Further, preexisting stable spines survived less following injury than controls, and such lost persistent spines were smaller in size than the surviving ones, which displayed long-term potentiation-like enlargement over weeks. These results suggest that peripheral nerve injury induces rapid and selective remodeling of cortical synapses, which is associated with neuropathic pain development, probably underlying, at least partially, long-lasting sensory changes in neuropathic subjects.

Possible involvement of tetrodotoxin-resistant sodium channels in cough reflex.

We examined the involvement of tetrodotoxin (TTX)-resistant sodium channels in the peripheral mechanisms of the cough reflex in mice. We also examined the possibility of using ambroxol as an effective antitussive agent, and found that it produced antitussive effects through the inhibition of TTX-resistant sodium channels. The inhalation of fenvalerate, at concentrations of 0.3, 1 and 3µg/ml, for 5min produced coughs in a concentration-dependent manner. Pretreatment with tetrodotoxin, at a dose of 1µg/kg, s.c., slightly but significantly reduced the number of fenvalerate (3µg/ml)-induced coughs. However, the number of fenvalerate-induced coughs in tetorodotoxin-treated mice was still significantly greater than those in vehicle (0.4% DMSO) alone inhaled mice. On the other hand, pretreatment with tetrodotoxin, at a dose of 1µg/kg, s.c., almost completely reduced the number of citric acid (0.25M)-induced coughs to the level in vehicle (saline) alone inhaled mice. Pretreatment with ambroxol, at doses of 10, 30, 100 and 300mg/kg, p.o., dose-dependently and significantly reduced the number of fenvalerate (3µg/ml)-induced coughs. The present findings indicate that TTX-resistant sodium channels may play an important role in the enhancement of C-fiber-mediated cough pathways. Furthermore, ambroxol may prove to be a useful cough suppressant.

Tetrodotoxin reduces cue-induced drug craving and anxiety in abstinent heroin addicts.

BACKGROUND: Tetrodotoxin (TTX) is a neurotoxin found in puffer fish and other marine animals. New clinical studies suggest that low-dose TTX can safely relieve severe, treatment-resistant cancer pain. The therapeutic potential of TTX in addiction is supported by studies in laboratory animals. The purpose of this double-blind, placebo-controlled study was to assess the effect of a single intramuscular dose of TTX on cue-induced craving and anxiety in abstinent heroin addicts. METHODS: Forty-five abstinent heroin addicts were randomly assigned to three treatment groups: placebo, 5 microg TTX, or 10 microg TTX. Participants were exposed to a neutral video or a heroin-related video. Craving, anxiety, blood pressure, and heart rate were measured pre- and post-exposure. RESULTS: Heroin-related cues increased both craving and anxiety and had no effect on blood pressure and heart rate. A single dose of TTX dose-dependently attenuated the increases in craving and anxiety while having no effect on blood pressure or heart rate. CONCLUSION: The results suggest that low-dose TTX is acutely effective in reducing cue-induced increases in heroin craving and associated anxiety.

Effect of TTX suppression of hippocampal activity following status epilepticus.

Status epilepticus (SE) is a severe neurological condition that can result in brain damage. In animals, SE is associated with cell loss and aberrant synaptogenesis. These pathological processes appear to be activity-dependent and may continue after the SE has ended. We postulated that suppression of electrical activity following SE at the site of the epileptic focus will reduce seizure-induced damage. To achieve this goal, tetrodotoxin (TTX) was used to suppress electrical activity in the hippocampi bilaterally following SE. Adult rats experienced lithium-pilocarpine-induced SE for 2h while controls underwent sham-SE with saline injections. Starting 12h after the SE or sham-SE rats received either continuous TTX (1 microM) or saline infusions through cannulas implanted in the bilateral hippocampi for 5h daily for 4 days. TTX resulted in significant EEG suppression and reduction in spikes and sharp waves. Rats were sacrificed 2 weeks after SE and the brains examined for cell loss and sprouting. Rats receiving TTX following SE had significantly more cell loss as well as a trend toward more mossy fiber sprouting than saline-treated rats following SE. TTX injection in sham-SE rats caused no cell loss or mossy fiber sprouting. These results suggest that suppression of electrical activity following SE is detrimental.

Rho kinase as a novel molecular therapeutic target for hypertensive internal anal sphincter.

BACKGROUND & AIMS: An increase in Rho kinase (ROK) activity has been associated with agonist-induced sustained contraction of the smooth muscle, but its role in the pathophysiology of spontaneously tonic smooth muscle is not known. METHODS: Present studies examined the effects of ROK inhibitor Y-27632 in the tonic smooth muscle of the rat internal anal sphincter (IAS) versus in the flanking phasic smooth muscle of the rectum. In addition, studies were performed to determine the relationship between the decreases in the basal IAS tone and the ROK activity. Confocal microscopic studies determined the cellular distribution of the smooth muscle-predominant isoform of ROK (ROCK-II) in the smooth muscle cells (SMCs). RESULTS: In in vitro studies using neurohumoral inhibitors and tetrodotoxin and the use of SMCs demonstrate direct relaxation of the IAS SMCs by Y-27632. The ROK inhibitor was more potent in the IAS than in the rectal smooth muscle. The IAS relaxation by Y-27632 correlated specifically with the decrease in ROK activity. Confocal microscopy revealed high levels of ROCK-II toward the periphery of the IAS SMCs. In in vivo studies, the lower doses of Y-27632 caused a potent and selective decrease in the IAS pressures without any adverse cardiovascular systemic effects. The ROK inhibitor also caused potent relaxation of the hypertensive IAS. CONCLUSIONS: RhoA/ROK play a crucial role in the maintenance of the basal tone in the IAS, and ROK inhibitors have a therapeutic potential in the IAS dysfunction characterized by the hypertensive IAS.

Neuropathic pain: early spontaneous afferent activity is the trigger.

Intractable neuropathic pain often results from nerve injury. One immediate event in damaged nerve is a sustained increase in spontaneous afferent activity, which has a well-established role in ongoing pain. Using two rat models of neuropathic pain, the CCI and SNI models, we show that local, temporary nerve blockade of this afferent activity permanently inhibits the subsequent development of both thermal hyperalgesia and mechanical allodynia. Timing is critical-the nerve blockade must last at least 3-5 days and is effective if started immediately after nerve injury, but not if started at 10 days after injury when neuropathic pain is already established. Effective nerve blockade also prevents subsequent development of spontaneous afferent activity measured electrophysiologically. Similar results were obtained in both pain models, and with two blockade methods (200mg of a depot form bupivacaine at the injury site, or perfusion of the injured nerve just proximal to the injury site with TTX). These results indicate that early spontaneous afferent fiber activity is the key trigger for the development of pain behaviors, and suggest that spontaneous activity may be required for many of the later changes in the sensory neurons, spinal cord, and brain observed in neuropathic pain models. Many pre-clinical and clinical studies of pre-emptive analgesia have used much shorter duration of blockade, or have not started immediately after the injury. Our results suggest that effective pre-emptive analgesia can be achieved only when nerve block is administered early after injury and lasts several days.

Mechanisms of atrial fibrillation termination by pure sodium channel blockade in an ionically-realistic mathematical model.

The mechanisms by which Na+-channel blocking antiarrhythmic drugs terminate atrial fibrillation (AF) remain unclear. Classical "leading-circle" theory suggests that Na+-channel blockade should, if anything, promote re-entry. We used an ionically-based mathematical model of vagotonic AF to evaluate the effects of applying pure Na+-current (I(Na)) inhibition during sustained arrhythmia. Under control conditions, AF was maintained by 1 or 2 dominant spiral waves, with fibrillatory propagation at critical levels of action potential duration (APD) dispersion. I(Na) inhibition terminated AF increasingly with increasing block, terminating all AF at 65% block. During 1:1 conduction, I(Na) inhibition reduced APD (by 13% at 4 Hz and 60% block), conduction velocity (by 37%), and re-entry wavelength (by 24%). During AF, I(Na) inhibition increased the size of primary rotors and reduced re-entry rate (eg, dominant frequency decreased by 33% at 60% I(Na) inhibition) while decreasing generation of secondary wavelets by wavebreak. Three mechanisms contributed to I(Na) block-induced AF termination in the model: (1) enlargement of the center of rotation beyond the capacity of the computational substrate; (2) decreased anchoring to functional obstacles, increasing meander and extinction at boundaries; and (3) reduction in the number of secondary wavelets that could provide new primary rotors. Optical mapping in isolated sheep hearts confirmed that tetrodotoxin dose-dependently terminates AF while producing effects qualitatively like those of I(Na) inhibition in the mathematical model. We conclude that pure INa inhibition terminates AF, producing activation changes consistent with previous clinical and experimental observations. These results provide insights into previously enigmatic mechanisms of class I antiarrhythmic drug-induced AF termination. The full text of this article is available online at http://circres.ahajournals.org

Chronic alterations in the cellular composition of spinal cord white matter following contusion injury.

Spinal cord injury (SCI) involves the loss of neurons and glia due to initial mechanical and secondary biochemical mechanisms. Treatment with the sodium channel blocker tetrodotoxin (TTX) reduces acute white matter pathology and increases both axon density and hindlimb function chronically at 6 weeks after injury. We investigated the cellular composition of residual white matter chronically to determine whether TTX also has a significant effect on the numbers and types of cells present. Rats received an incomplete thoracic contusion injury, in the presence or absence of TTX (0.15 nmole) injected focally, beginning at 15 min prior to injury. Six weeks later, cell density was significantly increased in the residual white matter of the dorsal, lateral, and ventral funiculi, both rostral and caudal to the injury site in both TTX-treated and injury control groups. Oligodendrocyte and astrocyte density was similar to normal but large numbers of cells expressing microglia/macrophage markers were present. Labeling with the progenitor markers nestin and NG2 showed that precursor cell density had also doubled or tripled as compared with uninjured controls. Some of these cells were also labeled for antigens that indicate their possible progression along an oligodendrocyte or astrocyte lineage. Our results support the hypothesis that the beneficial effect of TTX in SCI is related to its preservation of axons per se; no effect on chronic white matter cell composition was detected. They highlight the profound changes in cellular composition in preserved white matter chronically at 6 weeks after injury, including the accumulation of endogenous progenitor cells and the persistence of activated macrophages/microglia. The manipulation of these endogenous cells may be used in the future to enhance recovery after SCI.