Prolonged Duration Local Anesthesia by Combined Delivery of Capsaicin- and Tetrodotoxin-Loaded Liposomes.

BACKGROUND: Capsaicin, the active component of chili peppers, can produce sensory-selective peripheral nerve blockade. Coadministration of capsaicin and tetrodotoxin, a site-1 sodium channel blocker, can achieve a synergistic effect on duration of nerve blocks. However, capsaicin can be neurotoxic, and tetrodotoxin can cause systemic toxicity. We evaluated whether codelivery of capsaicin and tetrodotoxin liposomes can achieve prolonged local anesthesia without local or systemic toxicity. METHODS: Capsaicin- and tetrodotoxin-loaded liposomes were developed. Male Sprague-Dawley rats were injected at the sciatic nerve with free capsaicin, capsaicin liposomes, free tetrodotoxin, tetrodotoxin liposomes, and blank liposomes, singly or in combination. Sensory and motor nerve blocks were assessed by a modified hotplate test and a weight-bearing test, respectively. Local toxicity was assessed by histologic scoring of tissues at the injection sites and transmission electron microscopic examination of the sciatic nerves. Systemic toxicity was assessed by rates of contralateral nerve deficits and/or mortality. RESULTS: The combination of capsaicin liposomes and tetrodotoxin liposomes achieved a mean duration of sensory block of 18.2 hours (3.8 hours) [mean (SD)], far longer than that from capsaicin liposomes [0.4 hours (0.5 hours)] (P < .001) or tetrodotoxin liposomes [0.4 hours (0.7 hours)] (P < .001) given separately with or without the second drug in free solution. This combination caused minimal myotoxicity and muscle inflammation, and there were no changes in the percentage or diameter of unmyelinated axons. There was no systemic toxicity. CONCLUSIONS: The combination of encapsulated tetrodotoxin and capsaicin achieved marked prolongation of nerve block. This combination did not cause detectable local or systemic toxicity. Capsaicin may be useful for its synergistic effects on other formulations even when used in very small, safe quantities.

Polymer-tetrodotoxin conjugates to induce prolonged duration local anesthesia with minimal toxicity.

There is clinical and scientific interest in developing local anesthetics with prolonged durations of effect from single injections. The need for such is highlighted by the current opioid epidemic. Site 1 sodium channel blockers such as tetrodotoxin (TTX) are extremely potent, and can provide very long nerve blocks but the duration is limited by the associated systemic toxicity. Here we report a system where slow release of TTX conjugated to a biocompatible and biodegradable polymer, poly(triol dicarboxylic acid)-co-poly(ethylene glycol) (TDP), is achieved by hydrolysis of ester linkages. Nerve block by the released TTX is enhanced by administration in a carrier with chemical permeation enhancer (CPE) properties. TTX release can be adjusted by tuning the hydrophilicity of the TDP polymer backbone. In vivo, 1.0-80.0 µg of TTX released from these polymers produced a range of durations of nerve block, from several hours to 3 days, with minimal systemic or local toxicity.

Phototriggered Local Anesthesia.

We report a phototriggerable formulation enabling in vivo repeated and on-demand anesthesia with minimal toxicity. Gold nanorods (GNRs) that can convert near-infrared (NIR) light into heat were attached to liposomes (Lip-GNRs), enabling light-triggered phase transition of their lipid bilayers with a consequent release of payload. Lip-GNRs containing the site 1 sodium channel blocker tetrodotoxin and the α2-adrenergic agonist dexmedetomidine (Lip-GNR-TD) were injected subcutaneously in the rat footpad. Irradiation with an 808 nm continuous wave NIR laser produced on-demand and repeated infiltration anesthesia in the rat footpad in proportion to the irradiance, with minimal toxicity. The ability to achieve on-demand and repeated local anesthesia could be very beneficial in the management of pain.

Repeatable and adjustable on-demand sciatic nerve block with phototriggerable liposomes.

Pain management would be greatly enhanced by a formulation that would provide local anesthesia at the time desired by patients and with the desired intensity and duration. To this end, we have developed near-infrared (NIR) light-triggered liposomes to provide on-demand adjustable local anesthesia. The liposomes contained tetrodotoxin (TTX), which has ultrapotent local anesthetic properties. They were made photo-labile by encapsulation of a NIR-triggerable photosensitizer; irradiation at 730 nm led to peroxidation of liposomal lipids, allowing drug release. In vitro, 5.6% of TTX was released upon NIR irradiation, which could be repeated a second time. The formulations were not cytotoxic in cell culture. In vivo, injection of liposomes containing TTX and the photosensitizer caused an initial nerve block lasting 13.5 ± 3.1 h. Additional periods of nerve block could be induced by irradiation at 730 nm. The timing, intensity, and duration of nerve blockade could be controlled by adjusting the timing, irradiance, and duration of irradiation. Tissue reaction to this formulation and the associated irradiation was benign.

Tetrodotoxin-bupivacaine-epinephrine combinations for prolonged local anesthesia.

Currently available local anesthetics have analgesic durations in humans generally less than 12 hours. Prolonged-duration local anesthetics will be useful for postoperative analgesia. Previous studies showed that in rats, combinations of tetrodotoxin (TTX) with bupivacaine had supra-additive effects on sciatic block durations. In those studies, epinephrine combined with TTX prolonged blocks more than 10-fold, while reducing systemic toxicity. TTX, formulated as Tectin, is in phase III clinical trials as an injectable systemic analgesic for chronic cancer pain. Here, we examine dose-duration relationships and sciatic nerve histology following local nerve blocks with combinations of Tectin with bupivacaine 0.25% (2.5 mg/mL) solutions, with or without epinephrine 5 µg/mL (1:200,000) in rats. Percutaneous sciatic blockade was performed in Sprague-Dawley rats, and intensity and duration of sensory blockade was tested blindly with different Tectin-bupivacaine-epinephrine combinations. Between-group comparisons were analyzed using ANOVA and post-hoc Sidak tests. Nerves were examined blindly for signs of injury. Blocks containing bupivacaine 0.25% with Tectin 10 µM and epinephrine 5 µg/mL were prolonged by roughly 3-fold compared to blocks with bupivacaine 0.25% plain (P < 0.001) or bupivacaine 0.25% with epinephrine 5 µg/mL (P < 0.001). Nerve histology was benign for all groups. Combinations of Tectin in bupivacaine 0.25% with epinephrine 5 µg/mL appear promising for prolonged duration of local anesthesia.

Excitability and gap junction-mediated mechanisms in nucleus accumbens regulate self-stimulation reward in rats.

The nucleus accumbens (Acb) is a part of the striatum which integrates information from cortical and limbic brain structures, and mediates behaviors which reinforce reward. Previous work has suggested that neuronal synchrony mediated by gap junctions in Acb-related areas is involved in brain pleasure and reward. In order to gain insight into functional aspects of the neural information processing at the level of the striatum, we explored the possible role of Acb gap junctional communication and chemical synapses on reward self-stimulation in rats using positive reinforcement. Rats were trained to press a lever that caused an electrical current to be delivered into the hypothalamus, which is recognized to cause pleasure/reward. Intracerebral infusion into the Acb of the gap junctional blocker carbenoxolone (CBX) decreased the lever-pressing activity. Considering that the net effect of blocking gap junctions is a reduced synchronized output of the cellular activities, which at some level represents a decrease in excitability, two other inhibitors of neuronal excitability, carbamazepine (CBZ) and tetrodotoxin (TTX), were infused into the Acb and their effects on lever-pressing assessed. All manipulations that diminished excitability in the Acb resulted in reduced lever-pressing activity. CBX and TTX were also infused into motor cortex mediating forelimb lever-pressing with no effect. However, a manipulation that has the net effect of increasing excitation, the infusion of the opiate antagonist naloxone, also decreased significantly brain self-stimulation. We conclude that reward behaviors depend to a great extent on both excitability and gap junction-mediated mechanisms in Acb neuronal networks. Thus, the Acb provides a site for the study of pleasure/reward, addiction and conscious experience.

Tetrodotoxin for prolonged local anesthesia with minimal myotoxicity.

Conventional local anesthetics such as bupivacaine cause considerable myotoxicity and neurotoxicity, whereas tetrodotoxin (TTX) does not. Tetrodotoxin combined with bupivacaine or vasoconstrictors produces long-duration nerve blockade. To assess whether these prolonged blocks can be produced without increased myotoxicity, Sprague-Dawley rats were injected with bupivacaine, TTX, and both, or TTX plus epinephrine. Median durations of thermal nociceptive blockade were, respectively, 188, 401, 882, and 972 min. On dissection 4 days later, all tissues appeared macroscopically pristine. Muscle injury was at most mild to moderate in all animals, and the muscle injury scores for the combination formulations were not higher than for bupivacaine alone. Similarly, in differentiated cells from a myoblast cell line (C2C12), TTX caused either no or minimal worsening of cell viability from bupivacaine at 2 or 7 days. Epinephrine did not worsen TTX's relatively minimal cytotoxicity. Tetrodotoxin may thus be useful in producing prolonged nerve block with minimal myotoxicity and perhaps neurotoxicity.

Spontaneous retinal activity is tonic and does not drive tectal activity during activity-dependent refinement in regeneration.

During development, waves of activity periodically spread across retina to produce correlated activity that is thought to drive activity-dependent ordering in optic fibers. We asked whether similar waves of activity are produced in the retina of adult goldfish during activity-dependent refinement by regenerating optic fibers. Dual-electrode recordings of spontaneous activity were made at different distances across retina but revealed no evidence of retinal waves in normal retina or during regeneration. Retinal activity was tonic and lacked the episodic bursting associated with waves. Cross-correlation analysis showed that the correlated activity that was normally restricted to near neighbors (typically seen across 100-200 microm and absent at >500 microm) was not altered during regeneration. The only change associated with regeneration was a twofold reduction in ganglion cell firing rates. Because spontaneous retinal activity is known to be sufficient to generate refinement during regeneration in goldfish, we examined its effect on tectal activity. In normal fish, acutely eliminating retinal activity with TTX rapidly reduced tectal unit activity by >90%. Surprisingly, during refinement at 4-6 weeks, eliminating retinal activity had no detectable effect on tectal activity. Similar results were obtained in recordings from torus longitudinalis. After refinement at 3 months, tectal activity was again highly dependent on ongoing retinal activity. We conclude that spontaneous retinal activity drives tectal cells in normal fish and after regeneration but not during activity-dependent refinement. The implications of these results for the role of presynaptic activity in refinement are considered.

Auditory thalamus, dorsal hippocampus, basolateral amygdala, and perirhinal cortex role in the consolidation of conditioned freezing to context and to acoustic conditioned stimulus in the rat.

On the basis of previous experimental evidence, it is known that the auditory thalamus (AT), the dorsal hippocampus (DH), the basolateral amygdala (BLA), and the perirhinal cortex (PC) are involved in the mnemonic processing of conditioned freezing. In particular, BLA and PC appear to be involved both in conditioned stimulus (CS) and context conditioned freezing. Through AT, the auditory CS is sent to other sites, whereas DH is involved in context conditioning. Nevertheless, the existing evidence does not make it possible to assess AT, DH, BLA, and PC involvement during the consolidation phase of conditioned freezing. To address this question, fully reversible tetrodotoxin (TTX) inactivation was performed on adult male Wistar rats having undergone CS and context fear training. Anesthetized animals were injected stereotaxically with TTX (either 5 or 10 ng in 0.5 or 1.0 microliter of saline, according to site dimensions) at increasing post-acquisition delays. Context and CS freezing durations were measured during retention testing, always performed 48 and 72 hr after TTX administration. The results showed that AT inactivation does not disrupt consolidation of either contextual or auditory fear memories. In contrast, inactivation of the other three structures disrupted consolidation. For the DH, this disruption was specific to contextual cues and only occurred when inactivation was performed early (up to 1.5 hr) after training. The BLA and PC were shown to be involved in the consolidation of both contextual and auditory fear. Their involvement persisted for longer periods of time (2d for BLA and 8 d for PC). These findings provide information to build a temporal profile for the post-training processing of fear memories in structures known to be important for this form of learning. The results are discussed in relation to previous studies on conditioned freezing and other aversive conditioned response neural correlates.

Effect of activity blockade on changes in vibrissae-related patterns in the rat's primary somatosensory cortex induced by serotonin depletion.

Depletion of cortical serotonin (5-HT) during development results in a decrease in the size of the patches of thalamocortical afferents representing the mystacial vibrissae in lamina IV of the primary somatosensory cortex (SI). We previously suggested that this change may be due to a reduction in 5-HT-induced suppression of thalamocortical activity in these animals. The present experiments directly tested the role that modulation of activity may play in the morphologic changes observed after reducing cortical 5-HT concentrations. Serotonin was depleted from the cortex by systemic administration of 5,7-dihydroxytryptamine (5,7-DHT, 100 mg/kg) on the day of birth in animals that also had either tetrodotoxin (TTX)-impregnated or control implants placed unilaterally over the developing SI on this day. Other rat pups were treated with TTX-impregnated or control implants alone. Administration of 5,7-DHT reduced cortical serotonin levels and this effect was not significantly modified by the presence of either control or TTX-impregnated cortical implants. Administration of 5,7-DHT reduced the cross-sectional area of the cortical patches, demonstrated by acetylcholinesterase, corresponding to the vibrissae by 19.9% (P < 0.05). A similar reduction was observed in the animals treated with both 5,7-DHT and TTX-impregnated implants. Treatment with TTX-impregnated implants alone resulted in a 3.1% increase in patch size (P > 0.05). None of the treatments significantly altered the overall area of the part of SI devoted to the representation of the long mystacial vibrissae. These results suggest that the effects of 5-HT depletion on the size of the cortical patches representing the long vibrissae are independent of activity that can be blocked by administration of TTX.

A re-examination of tetrodotoxin for prolonged duration local anesthesia.

BACKGROUND: Highly potent toxins such as tetrodotoxin that block sodium channels with great specificity have been studied for many years and can provide prolonged blockade when coadministered with vasoconstrictors or conventional local anesthetics. Their utility has been constrained, however, by systemic toxicity. The authors examined the efficacy of tetrodotoxin with and without epinephrine or bupivacaine for producing prolonged-duration sciatic nerve blockade in the rat, and they assessed the degree of concomitant toxicity. METHODS: Rats received percutaneous sciatic nerve blockade using tetrodotoxin with and without epinephrine or bupivacaine. A subset received subcutaneous injections at the nuchal midline. Nociceptive, proprioceptive, and motor blockade were quantified using contralateral leg responses as controls for systemic effects. RESULTS: Tetrodotoxin without epinephrine produced sciatic nerve blockade, but with considerable toxicity at most effective doses. Epinephrine reduced the median effective concentration of tetrodotoxin for nociception from 37.6 to 11.5 microM and prolonged its duration, such that reversible blocks lasting > 13 h were achieved. Epinephrine reduced measures of systemic distribution and increased the median lethal dose of tetrodotoxin from 40 to 53.6 nmole/kg, thus more than quadrupling the therapeutic index. Bupivacaine increased the local anesthetic potency of tetrodotoxin, reduced its systemic toxicity, and, when coinjected subcutaneously, increased the median lethal dose from 43.7 to 47.7 nmole/kg. The addition of epinephrine did not further improve the effectiveness of the bupivacaine-tetrodotoxin combination. CONCLUSION: Combinations of epinephrine or bupivacaine with tetrodotoxin or with other high-potency toxins active on sodium channels should be examined for the potential to provide clinically useful, prolonged nerve blockade.

Experimental study of tetrodotoxin, a long-acting topical anesthetic.

PURPOSE: To determine the effectiveness and toxicity of tetrodotoxin for use as a long-acting topical anesthetic. METHODS: Four groups of six rabbits each received a 40-microl aliquot of either tetrodotoxin in one of three concentrations (10 mM, 1 mM, or 0.1 mM) or proparacaine 0.5% into the inferior conjunctival cul-de-sac of one eye, with the fellow eye of each rabbit receiving 40 microl of a 60-mM, pH 4.3 sodium citrate vehicle as a control. Corneal sensation was tested for up to 8 hours after administration of drugs, and response was noted by no blink, partial blink without full eyelid closure, and full blink. Slit-lamp examination at 12 and 24 hours after administration and pachymetry before and 24 hours after administration were performed to detect corneal toxicity. RESULTS: Rabbits receiving all three concentrations of tetrodotoxin did not demonstrate any ocular irritation, corneal thickening, or signs of systemic toxicity. At a dose of 10 mM, tetrodotoxin produced an anesthetic effect lasting up to 8 hours. At 1 mM, tetrodotoxin was an effective but shorter-acting anesthetic. At 0.1 mM, tetrodotoxin had no significant anesthetic effect. Proparacaine-treated rabbits initially were anesthetic, but this effect was largely gone by 1 hour and completely gone by 3 hours. CONCLUSIONS: Tetrodotoxin is a long-acting topical anesthetic in the rabbit cornea. Although additional toxicity studies are required, tetrodotoxin may provide an effective, long-lasting topical anesthetic for use in pain control after corneal procedures such as photorefractive keratectomy.

Experimental use of tetrodotoxin for corneal pain after excimer laser keratectomy.

PURPOSE: To determine the duration of anesthesia, effect on corneal reepithelialization, and systemic toxicity of topical tetrodotoxin (TTX) administered after excimer laser keratectomy. METHODS: Two groups of six rabbits each underwent excimer laser keratectomy in the right eye to create a 5-mm-diameter wound, 75 mm in depth. One group then received a 40-microl aliquot of topical 1 mM TTX into the injured eye, whereas the other group received 40 microl of the sodium citrate vehicle as a control. The rabbits were treated with TTX or vehicle again at 6, 12, 18, and 24 h. Corneal sensation was tested at 3, 6, 9, 12, 15, 18, 21, 24, 30, 32, and 40 h. To determine whether TTX inhibited corneal reepithelialization, compared with vehicle-treated control eyes, the healing rate of the epithelial defect was measured. RESULTS: Administration of TTX every 6 h for 24 h produced nearly complete anesthesia for > or = 30 h. At 32 h, 8 h after the final application of TTX, there was still significant anesthesia of the TTX-treated corneas (p = 0.0325, Wilcoxon test). Normal corneal sensation in all TTX-treated animals returned at 40 h, or 16 h after the final dose. In contrast, vehicle-treated eyes all had normal sensation for nearly the entire duration of the experiment. At 40 h, the TTX-treated eyes had slightly larger defects than vehicle-treated eyes, 7.85+/-1.74 versus 4.54+/-1.31 mm2 (p < 0.025, t test). However, at 49 h and thereafter, both groups were equally healed (p > 0.05, t test). No systemic toxicity was observed in any of the rabbits. CONCLUSION: Topical TTX is a long-acting and nontoxic local anesthetic in a rabbit model of excimer laser keratectomy.

Electrical stimulation activates two different sites within the guinea pig cochlea.

This study investigates whether auditory brainstem responses (ABRs) can be used to assess the functioning of electrically stimulated cochleas. Electrically evoked auditory brainstem responses (EABRs) were recorded in guinea pigs with normal hearing and guinea pigs deafened by amikacin, a powerful ototoxic antibiotic, combined with diuretic aminooxyacetic acid (AOAA). Two different types of EABRs were observed in normal animals, depending on the electrical pulse intensity applied to the round window: long-latency brainstem responses were evoked by low stimulation intensities, short-latency brainstem responses by high intensities. The absence of effect of strychnine applied intracochlearly ruled out the possibility of medial efferents being involved in these responses. Conversely, an intracochlear application of tetrodotoxin (TTX), an Na(+)-channel blocker, resulted in the disappearance of both types of responses, attesting that the sites activated by the electrical stimulation were located within the cochlea. In AOAA/ amikacin poisoned cochleas, in which most of the hair cells were missing with apparently normal ganglion neurons, the long-latency brainstem responses evoked by low intensities were completely lacking. These findings suggest that low currents applied to the round window of the guinea pig cochlea primarily activate the hair cells, the neurons being directly excited at higher intensities.

Effects of postnatal blockage of cortical activity with tetrodotoxin upon lesion-induced reorganization of vibrissae-related patterns in the somatosensory cortex of rat.

Previous studies have shown that postnatal blockade of thalamocortical activity with either tetrodotoxin (TTX) or the NMDA receptor antagonist DL-2-amino-5-phosphonovalerate (APV) does not prevent the formation of vibrissae-related patterns. In the present study, blockade of cortical activity with TTX was combined with ablation of a row of vibrissae follicles or transection of the infraorbital nerve (ION, the trigeminal nerve branch that supplies the vibrissae follicles) to determine whether the cortical reorganization that follows these lesions in otherwise untreated animals was dependent upon neuronal activity that could be blocked with TTX. The results demonstrated that cortical TTX implants had no quantitative or qualitative effects upon the cortical reorganization that followed either vibrissae follicle cauterization or ION transection.

Peripheral generators of the vestibular evoked potentials (VsEPs) in the chick.

Electrophysiological activity in response to linear acceleration stimuli was recorded from young chickens by means of subcutaneous electrodes. This investigation had 2 purposes: (1) to establish the vestibular origin of the potentials; and (2) to investigate the contribution of each vestibular labyrinth to the response. The stimuli consisted of pulses of linear acceleration delivered by a mechanical vibrator (shaker). In the first set of experiments vestibular evoked potentials (VsEPs) were recorded prior to and 24 h after bilateral cochlea removal. In the second set of experiments responses were recorded before and after unilateral or bilateral intralabyrinthine injections of tetrodotoxin (TTX). Different groups of subjects were used for each experimental condition. The general morphology of the VsEPs was maintained after bilateral cochlea removal. Absolute latency of wave P2, the most prominent component of the response, was not significantly affected by the manipulation. Unilateral intralabyrinthine TTX injections consistently prolonged the latency and reduced the amplitude of wave P2. Following binaural TTX injections we were unable to elicit responses at the acceleration levels used in this study. The results from these experiments suggest that: (1) the activity recorded in response to linear acceleration stimuli is vestibular in origin; (2) when recorded from intact animals the evoked response is composed of activity from both vestibular systems; and (3) TTX consistently blocks the activity of the vestibular portion of the VIIIth cranial nerve.