Interactions of peripheral endothelin-1 and nerve growth factor as contributors to persistent cutaneous pain.

Endothelin-1 (ET-1) and Nerve Growth Factor (NGF) are proteins, released from cancer-ridden tissues, which cause spontaneous pain and hypersensitivity to noxious stimuli. Here we examined the electrophysiological and behavioral effects of these two agents for evidence of their interactions. Individual small-medium cultured DRG sensory neurons responded to both ET-1 (50 nM, n=6) and NGF (100 ng/ml, n=4), with increased numbers of action potentials and decreased slow K(+) currents; pre-exposure to ET-1 potentiated NGF´s actions, but not vice versa. Behaviorally, single intraplantar (i.pl.) injection of low doses of ET-1 (20 pmol) or NGF (100 ng), did not increase hindpaw tactile or thermal sensitivity, but their simultaneous injections sensitized the paw to both modalities. Daily i.pl. injections of low ET-1 doses in male rats caused tactile sensitization after 21 days, and enabled further tactile and thermal sensitization from low dose NGF, in ipsilateral and contralateral hindpaws. Single injections of 100 ng NGF, without changing the paw's tactile sensitivity by itself, acutely sensitized the ipsilateral paw to subsequent injections of low ET-1. The sensitization from repeated low ET-1 dosing and the cross-sensitization between NGF and ET-1 were both significantly greater in female than in male rats. These findings reveal a synergistic interaction between cutaneously administered low doses of NGF and ET-1, which could contribute to cancer-related pain.

Bupivacaine inhibits endothelin-1-evoked increases in intracellular calcium in model sensory neurons.

BACKGROUND: Endothelin-1 (ET-1) induces pain-like behavior in animals and man by activating the Gq protein-coupled receptor endothelin-A (ETA ). Activation of ETA receptors on nociceptor membranes evokes intracellular calcium transients and alters membrane Na(+) and K(+) channel and TRPV1 currents, leading to neuronal hyper-excitability manifested by spontaneous and evoked pain behaviors in vivo. In addition to blocking sodium channels, local anesthetics inhibit the Gq protein-coupled signaling of several inflammatory and pro-algesic mediators. In this study, we aimed to investigate the actions of local anesthetics on ETA -mediated increases in intracellular calcium in ND7/104 model sensory neurons. METHODS: Increases in intracellular calcium were measured by the fluorescent indicator fura-2 in a sensory neuron-derived cell line (ND7/104), which endogenously expresses ETA receptors. Effects of lidocaine and bupivacaine, along with their respective membrane-impermeant derivatives QX-314, LEA-123 and LEA-124, on peak calcium responses to ET-1 were measured. RESULTS: Bupivacaine suppressed ET-1 responses in a concentration-dependent and non-competitive manner with an IC50 of 3.79 ± 1.63 mM. Bupivacaine (6 mM) reduced the Emax for ET-1 from 50.07 ± 1.91 mM to 27.30 ± 2.92 mM. The actions of bupivacaine occurred quickly and were rapidly reversible. Membrane-impermeant analogs of bupivacaine (LEA-123 and LEA-124, 6 mM) were without effect, as was lidocaine (10 mM) and its quaternary derivative QX-314 (10 mM). CONCLUSION: Bupivacaine inhibits ETA -mediated calcium transients at clinically relevant concentrations through an intracellular target. The anti-inflammatory and analgesic actions of bupivacaine may be at least partially due to its inhibitory action on Gq -coupled receptors, including ETA.

The p75NTR signaling cascade mediates mechanical hyperalgesia induced by nerve growth factor injected into the rat hind paw.

Nerve growth factor (NGF) augments the excitability of isolated rat sensory neurons through activation of the p75 neurotrophin receptor (p75(NTR)) and its downstream sphingomyelin signaling cascade, wherein neutral sphingomyelinase(s) (nSMase), ceramide, and the atypical protein-kinase C (aPKC), protein-kinase M zeta (PKMζ), are key mediators. Here we examined these same receptor-pathways in vivo for their role in mechanical hyperalgesia from exogenous NGF. Mechanical sensitivity was tested by the number of paw withdrawals in response to 10 stimuli (PWF=n/10) by a 4-g von Frey hair (VFH, testing "allodynia") and by 10 and 15g VFHs (testing "hyperalgesia"). NGF (500ng/10µL) injected into the male rat's plantar hind paw induced long-lasting ipsilateral mechanical hypersensitivity. Mechano-hypersensitivity, relative to baseline responses and to those of the contralateral paw, developed by 0.5-1.5h and remained elevated at least for 21-24h, Acute intraplantar pre-treatment with nSMase inhibitors, glutathione (GSH) or GW4869, prevented the acute hyperalgesia from NGF (at 1.5h) but not that at 24h. A single injection of N-acetyl sphingosine (C2-ceramide), simulating the ceramide produced by nSMase activity, induced ipsilateral allodynia that persisted for 24h, and transient hyperalgesia that resolved by 2h. Intraplantar injection of hydrolysis-resistant mPro-NGF, selective for the p75(NTR) over the tyrosine kinase (TrkA) receptor, gave very similar results to NGF and was susceptible to the same inhibitors. Hyperalgesia from both NGF and mPro-NGF was prevented by paw pre-injection with blocking antibodies to rat p75(NTR) receptor. Finally, intraplantar (1day before NGF) injection of mPSI, the myristolated pseudosubstrate inhibitor of PKCζ/PKMζ, decreased the hyperalgesia resulting from NGF or C2-ceramide, although scrambled mPSI was ineffective. The findings indicate that mechano-hypersensitivity from peripheral NGF involves the sphingomyelin signaling cascade activated via p75(NTR), and that a peripheral aPKC is essential for this sensitization.

State-Dependent Inhibition of Sodium Channels by Local Anesthetics: A 40-Year Evolution.

Knowledge about the mechanism of impulse blockade by local anesthetics has evolved over the past four decades, from the realization that Na+ channels were inhibited to affect the impulse blockade to an identification of the amino acid residues within the Na+ channel that bind the local anesthetic molecule. Within this period appreciation has grown of the state-dependent nature of channel inhibition, with rapid binding and unbinding at relatively high affinity to the open state, and weaker binding to the closed resting state. Slow binding of high affinity for the inactivated state accounts for the salutary therapeutic as well as the toxic actions of diverse class I anti-arrhythmic agents, but may have little importance for impulse blockade, which requires concentrations high enough to block the resting state. At the molecular level, residues on the S6 transmembrane segments in three of the homologous domains of the channel appear to contribute to the binding of local anesthetics, with some contribution also from parts of the selectivity filter. Binding to the inactivated state, and perhaps the open state, involves some residues that are not identical to those that bind these drugs in the resting state, suggesting spatial flexibility in the "binding site". Questions remaining include the mechanism that links local anesthetic binding with the inhibition of gating charge movements, and the molecular nature of the theoretical "hydrophobic pathway" that may be critical for determining the recovery rates from blockade of closed channels, and thus account for both therapeutic and cardiotoxic actions.

Contralateral paw sensitization following injection of endothelin-1: effects of local anesthetics differentiate peripheral and central processes.

Subcutaneous injection of the peptide endothelin-1 (ET-1) into the rat's footpad is known to cause rapid, transient ipsilateral mechanical and thermal sensitization and nocifensive hind paw flinching. Here we report that local injection of ET-1 (2 nmoles) into one hind paw slowly sensitizes the contralateral paw to chemical and mechanical stimulation. There was a 1.5-2-fold increase in the hind paw flinching response, over that from the first injection, to a second injection of the same dose of ET-1 delivered 24 h later into the contralateral paw. A similar increase in the number of flinches during the second phase of the response to formalin also occurred in the contralateral paw 24 h after ET-1. The contralateral paw withdrawal threshold to von Frey hairs was lowered by approximately 55% at 24 h after ipsilateral ET-1 injection. ET-1 injected s.c. at a segmentally unrelated location, the nuchal midline, caused no sensitization of the paws, obviating a systemic route of action. Local anesthetic block of the ipsilateral sciatic nerve during the period of initial response to ipsilateral ET-1 prevented contralateral sensitization, indicating the importance of local afferent transmission, although ipsilateral desensitization was not changed. These findings suggest that peripheral ET-1 actions lead to central sensitization that alters responses to selected stimuli.

Novel ideas of local anaesthetic actions on various ion channels to ameliorate postoperative pain.

This review considers the ion channels that underlie transduction of nociceptive energies in the periphery, that are involved in impulse initiation and propagation in peripheral sensory neurones, and that participate in pre- and post-synaptic actions in the spinal cord dorsal horn, in light of their susceptibility to local anaesthetics. Although there are results from experiments on isolated cells and tissues ex vivo that support the hypothesized actions, their extrapolation to actions in vivo and the consequences for peri- and postoperative pain control are largely speculative.

Local anesthetics.

Local anesthetics are used broadly to prevent or reverse acute pain and treat symptoms of chronic pain. This chapter, on the analgesic aspects of local anesthetics, reviews their broad actions that affect many different molecular targets and disrupt their functions in pain processing. Application of local anesthetics to peripheral nerve primarily results in the blockade of propagating action potentials, through their inhibition of voltage-gated sodium channels. Such inhibition results from drug binding at a site in the channel's inner pore, accessible from the cytoplasmic opening. Binding of drug molecules to these channels depends on their conformation, with the drugs generally having a higher affinity for the open and inactivated channel states that are induced by membrane depolarization. As a result, the effective potency of these drugs for blocking impulses increases during high-frequency repetitive firing and also under slow depolarization, such as occurs at a region of nerve injury, which is often the locus for generation of abnormal, pain-related ectopic impulses. At distal and central terminals the inhibition of voltage-gated calcium channels by local anesthetics will suppress neurogenic inflammation and the release of neurotransmitters. Actions on receptors that contribute to nociceptive transduction, such as TRPV1 and the bradykinin B2 receptor, provide an independent mode of analgesia. In the spinal cord, where local anesthetics are present during epidural or intrathecal anesthesia, inhibition of inotropic receptors, such as those for glutamate, by local anesthetics further interferes with neuronal transmission. Activation of spinal cord mitogen-activated protein (MAP) kinases, which are essential for the hyperalgesia following injury or incision and occur in both neurons and glia, is inhibited by spinal local anesthetics. Many G protein-coupled receptors are susceptible to local anesthetics, with particular sensitivity of those coupled via the Gq alpha-subunit. Local anesthetics are also infused intravenously to yield plasma concentrations far below those that block normal action potentials, yet that are frequently effective at reversing neuropathic pain. Thus, local anesthetics modify a variety of neuronal membrane channels and receptors, leading to what is probably a synergistic mixture of analgesic mechanisms to achieve effective clinical analgesia.

Preferential block of small myelinated sensory and motor fibers by lidocaine: in vivo electrophysiology in the rat sciatic nerve.

BACKGROUND: Controversy still surrounds the differential susceptibility of nerve fibers to local anesthetics and its relation to selective functional deficits. In the current study we report features of conduction blockade in different classes of rat sciatic nerve fibers after injection of lidocaine by a percutaneous procedure that closely resembles clinical applications. METHODS: In 30 adult male Sprague-Dawley rats (weight, 300-400 g) during general anesthesia, impulses were recorded in different classes of sensory axons (large, Aalpha and beta fibers; small, Adelta myelinated fibers and unmyelinated C fibers) and motor axons (large, Aalpha fibers; small, Agamma myelinated fibers) classified by conduction velocity. The sciatic nerve was stimulated distally, and impulses were recorded from small filaments teased from L4-L5 dorsal (sensory) and ventral (motor) roots sectioned acutely from the spinal cord. Lidocaine at concentration of 0.05-1% was injected percutaneously in 0.1-ml solutions at the sciatic notch. Both tonic (stimulated at 0.5 Hz) and use-dependent (stimulated at 40 Hz for Adelta and Agamma fibers and at 5 Hz for C fibers) impulse inhibitions by lidocaine were assayed. RESULTS: Minimal effective (threshold) lidocaine concentrations (i.e., to block conduction in 10% of fibers) were, for sensory, 0.03% for Adelta, 0.07% for Aalphabeta, and 0.09-0.1% for C fibers, and for motor, 0.03% for Agamma and 0.05% for Aalpha fibers. The order of fiber susceptibility, ranked by concentrations that gave peak tonic fiber blockade of 50% (IC50s), was Agamma > Adelta = Aalpha > Aalphabeta > C. Faster-conducting C fibers (conduction velocity > 1 m/s) were more susceptible (IC50 = 0.13%) than slower ones (conduction velocity < 1 m/s; IC50 = 0.30%). At 1% lidocaine, all fibers were tonically blocked. Use-dependent effects accounted for only a modest potentiation of block (at a lidocaine concentration of 0.25%) in Adelta and Agamma fibers, and in C fibers phasic stimulation had even smaller effects and sometimes relieved tonic block. CONCLUSIONS: Susceptibility to lidocaine does not strictly follow the "size principle" that smaller (slower) axons are always blocked first. This order of fiber blockade is qualitatively consistent with previous reports of the order of functional deficits in the rat after percutaneous lidocaine, that is, motor = proprioception > nociception, if we assume that motor deficits first arise from conduction failure in Agamma fibers and that nociception relies on C fiber conduction.

Development of a novel probe for measuring drug binding to the F1*S variant of human alpha 1-acid glycoprotein.

A novel probe was developed to measure drug association with the F1*S variant of the human serum protein alpha 1-acid glycoprotein (AGP). The molecule 2-hydroxy-3,5-diiodo-N-[2(diethylamino)ethyl]benzamide (DEDIC) binds to AGP, quenching its native fluorescence. This quenching was fitted to a two-site model giving apparent dissociation constants of 0.049 +/- 0.005 and 12 +/- 2 microM (mean +/- SEM). Quenching of each of the separate variants of AGP by DEDIC was itself described by a two-site model, giving for the F1*S variant K(D)(1)((F1*S)) = 0.041 +/- 0.010 microM and K(D)(2)((F1*S)) = 29 +/- 7 microM; and for the A variant K(D)(1)((A)) = 0.31 +/- 0.18 microM and K(D)(2)((A)) = 8.8 +/- 0.7 microM. The utility of DEDIC in probing drug interactions with isolated variants was demonstrated in competition experiments with the model drugs amitriptyline and bupivacaine. In addition, the selectivity of DEDIC for variant F1*S rendered it capable of probing the binding of drugs (including the variant A-selective drug amitriptyline) to F1*S in a mixture of variants, such as occurs naturally in whole AGP. DEDIC is unique as an F1*S variant-selective probe of drug binding to whole AGP that is also sufficiently soluble to serve as a probe of drug binding to the lower affinity sites on isolated A and F1*S variants.

Endothelin-1 activates ET(A) receptors to increase intracellular calcium in model sensory neurons.

Endothelin-1 (ET-1) induces endothelin-A (ETA) receptor-mediated pain and selective excitation of nociceptors. Here we studied ET-1-induced changes in intracellular calcium (Ca2+in) in Fura-2 loaded mouse neuroblastoma-rat dorsal root ganglion hybrid cells (ND7/104). ET-1 (1-400 nM) induced concentration-dependent, transient increases in Ca2+in, probably of intracellular source. Responses to repeated application declined with increasing ET-1 concentration, implying receptor desensitization. Treatment of cells with the selective ETA receptor antagonist, BQ-123, produced a dose-dependent inhibition of the response that was 20% of ET-1 alone (IC50 = 20 nM, KI = 7 nM). No inhibition of the calcium response was observed with the selective ETB antagonist, BQ-788 (10-1000 nM). These results demonstrate that ET-1 induces dose- and ETA receptor-dependent release of Ca2+in in nociceptor-like neurons, and permit further examination of the pathways that underlie ET-1-induced pain signaling.

Local injection of endothelin-1 produces pain-like behavior and excitation of nociceptors in rats.

Neurobehavioral and neurophysiological actions of the peptide endothelin-1 (ET-1) were investigated after subcutaneous plantar hindpaw injections in adult male Sprague Dawley rats. Hindpaw flinching developed within minutes after ET-1 (8-16 nmol) injection, peaked at 30 min, lasted for 60 min, and was strongly inhibited by the endothelin-A (ET(A)) receptor antagonist, BQ-123 (3.2 m). In separate experiments, impulse activity of single, physiologically characterized sensory C-, Adelta-, and Abeta-fibers was recorded from the sciatic nerve in anesthetized rats after subcutaneous injections of endothelin-1 (1-20 nmol), alone or together with BQ-123 (3.2 m), into the plantar hindpaw receptive fields of these units. All nociceptive C-fibers (31 of 33 C-fibers studied) were excited by ET-1 (1-20 nmol) in a dose-dependent manner. For doses of 16-20 nmol, the mean latency for afferent activation after injection of ET-1 was 3.16 +/- 0.31 min, and the mean and maximum response frequency were 2.02 +/- 0.48 impulses (imp)/sec and 14.0 +/- 3.2 imp/sec, respectively. All 10 nociceptive Adelta-fibers (of 12 Adelta-fibers studied) also responded to 1-20 nmol of ET-1 in a dose-dependent manner with a mean latency of 3.5 +/- 0.12 min and mean response frequency of 3.3 +/- 2.3 imp/sec. In contrast, most Abeta-fibers (9 of 12) did not respond to ET-1. BQ-123, when coinjected with ET-1, blocked ET-1-induced activation in all C- and Adelta-fibers tested. These data demonstrate that subcutaneous administration of ET-1 to the rat plantar hindpaw produces pain-like behavior and selective excitation of nociceptive fibers through activation of ET(A) receptors.

Lidocaine selectively blocks abnormal impulses arising from noninactivating Na channels.

Abnormal, repetitive impulse firing arising from incomplete inactivation of Na+ channels may be involved in several diseases of muscle and nerve, including familial myotonias and neuropathic pain syndromes. Systemic local anesthetics have been shown to have clinical efficacy against myotonias and some forms of neuropathic pain, so we sought to develop an in vitro model to examine the cellular basis for these drugs' effects. In frog sciatic nerves, studied in vitro by the sucrose-gap method, peptide alpha-toxins from sea anemone (ATXII) or scorpion (LQIIa) venom, which inhibit Na+ channel inactivation, induced repetitively firing compound action potentials (CAPs) superimposed on a plateau depolarization lasting several seconds. The initial spike of the CAP was unaffected, but the plateau and repetitive firing were strongly suppressed by 5-30 microM lidocaine. Lidocaine caused a rapid, concentration-dependent decay of the plateau, quantitatively consistent with blockade of open Na(+) channels. Early and late repetitive firing were equally suppressed by lidocaine with IC50 = 10 microM. After washout of lidocaine and LQIIa, the plateau and repetitive firing remained for > 1 h, showing that lidocaine had not caused dissociation of channel-bound alpha-toxin. These findings indicate that therapeutic concentrations of lidocaine can reverse the "abnormal" features of action potentials caused by non-inactivating Na+ channels without affecting the normal spike component.

Block of human heart hH1 sodium channels by the enantiomers of bupivacaine.

BACKGROUND: S(-)-bupivacaine reportedly exhibits lower cardiotoxicity but similar local anesthetic potency compared with R(+)-bupivacaine. The bupivacaine binding site in human heart (hH1) Na+ channels has not been studied to date. The authors investigated the interaction of bupivacaine enantiomers with hH1 Na+ channels, assessed the contribution of putatively relevant residues to binding, and compared the intrinsic affinities to another isoform, the rat skeletal muscle (mu1) Na+ channel. METHODS: Human heart and mu1 Na+ channel alpha subunits were transiently expressed in HEK293t cells and investigated during whole cell voltage-clamp conditions. Using site-directed mutagenesis, the authors created point mutations at positions hH1-F1760, hH1-N1765, hH1-Y1767, and hH1-N406 by introducing the positively charged lysine (K) or the negatively charged aspartic acid (D) and studied their influence on state-dependent block by bupivacaine enantiomers. RESULTS: Inactivated hH1 Na+ channels displayed a weak stereoselectivity with a stereopotency ratio (+/-) of 1.5. In mutations hH1-F1760K and hH1-N1765K, bupivacaine affinity of inactivated channels was reduced by approximately 20- to 40-fold, in mutation hH1-N406K by approximately sevenfold, and in mutations hH1-Y1767K and hH1-Y1767D by approximately twofold to threefold. Changes in recovery of inactivated mutant channels from block paralleled those of inactivated channel affinity. Inactivated hH1 Na+ channels exhibited a slightly higher intrinsic affinity than mu1 Na+ channels. CONCLUSIONS: Differences in bupivacaine stereoselectivity and intrinsic affinity between hH1 and mu1 Na+ channels are small and most likely of minor clinical relevance. Amino acid residues in positions hH1-F1760, hH1-N1765, and hH1-N406 may contribute to binding of bupivacaine enantiomers in hH1 Na+ channels, whereas the role of hH1-Y1767 remains unclear.

Addition of sodium bicarbonate to lidocaine decreases the duration of peripheral nerve block in the rat.

BACKGROUND: Adding sodium bicarbonate to lidocaine to enhance its efficacy during peripheral nerve block is controversial. The authors studied the effect of adding sodium bicarbonate to lidocaine with and without epinephrine versus equivalent alkalinization by sodium hydroxide (NaOH) on onset, degree, and duration of peripheral nerve block. METHODS: Part I examined alkalinization by sodium bicarbonate versus NaOH to pH 7.8 on 0.5% lidocaine, with and without epinephrine (1:100,000), prepared from crystalline salt. Part II examined 0.5% and 1.0% commercial lidocaine solutions, with and without epinephrine, either unalkalinized or alkalinized with sodium bicarbonate or NaOH. With NaOH, pH was adjusted to 7.8, but with sodium bicarbonate, no pH adjustments were made to simulate clinical conditions. RESULTS: In part I, addition of either NaOH or sodium bicarbonate to 0.5% lidocaine without epinephrine produced a faster onset than did unalkalinized lidocaine, without effecting degree or duration of block. In solutions with epinephrine there were no differences in onset, degree, or duration between lidocaine alkalinized with sodium bicarbonate versus NaOH. In part II, addition of sodium bicarbonate or NaOH to 1.0% commercial lidocaine without epinephrine did not accelerate onset compared with the unalkalinized solution. However, adding sodium bicarbonate decreased the degree and duration of block by 25% and more than 50%, respectively, compared with lidocaine unalkalinized and alkalinized with NaOH. With epinephrine, sodium bicarbonate hastened onset without effecting degree and duration compared with the unalkalinized solution. CONCLUSIONS: With 1% commercial lidocaine without epinephrine, sodium bicarbonate decreases the degree and duration of block. However, in solutions with epinephrine, sodium bicarbonate hastens onset, without effecting degree or duration.

Potency of bupivacaine stereoisomers tested in vitro and in vivo: biochemical, electrophysiological, and neurobehavioral studies.

BACKGROUND: Chiral local anesthetics, such as ropivacaine and levobupivacaine, have the potential advantage over racemic mixtures in showing reduced toxic side effects. However, these S-(levo, or "-")isomers also have reportedly lower potency than their optical antipode, possibly resulting in no advantage in therapeutic index. Potency for local anesthetics inhibiting Na+ channels or action potentials depends on the pattern of membrane potential and so also does the stereopotency ratio. Here the authors have quantitated the stereopotencies of R-, S-, and racemic bupivacaine, comparing several in vitro assays of neuronal Na+ channels with those from in vivo functional nerve block, to establish relative potencies and to understand better the role of different modes of channel inhibition in overall functional anesthesia. METHODS: The binding of bupivacaine to Na+ channels was assessed indirectly by its antagonism of [3H]-batrachotoxin binding to rat brain synaptosomes. Inhibition of Na+ currents by bupivacaine was directly assayed in voltage-clamped GH-3 neuroendocrine cells. Neurobehavioral functions were disrupted by bupivacaine percutaneously injected (0.1 ml; 0.0625-1.0%) at the rat sciatic nerve and semiquantitatively assayed. Concentration-dependent actions of R-, S-, and racemic bupivacaine were compared for their magnitude and duration of action. RESULTS: Competitive batrachotoxin displacement has a stereopotency ratio of R:S = 3:1. Inhibition of Na+ currents with different prepulse potentials shows that S > R potency when the membrane is hyperpolarized, and R > S potency when it is depolarized from normal resting values. Functional deficits assayed in vivo usually demonstrate no consistent enantioselectivity and only a modest stereopotency (R:S = 1.2-1.3) for peak analgesia achieved at the lowest doses. Other functions display no significant stereopotency in either the degree, the duration, or their product (area under the curve) at any dose. CONCLUSION: Although the in vitro actions of bupivacaine showed stereoselectivity ratios of 1.3-3:1 (R:S), in vivo nerve block at clinically used concentrations showed much smaller ratios for peak effect and no significant enantioselectivity for duration. A primary role for the blockade of resting rather than open or inactivated Na+ channels may explain the modest stereoselectivity in vivo, although stereoselective factors controlling local disposition cannot be ruled out. Levo-(S-)bupivacaine is effectively equipotent to R- or racemic bupivacaine in vivo for rat sciatic nerve block.

The addition of dilute epinephrine produces equieffectiveness of bupivacaine enantiomers for cutaneous analgesia in the rat.

UNLABELLED: We investigated the effectiveness for cutaneous analgesia of bupivacaine (Bup) stereoisomers in male rats. As a model of infiltration anesthesia, inhibition of a nocifensive reflex by subcutaneous injection of 0.6 mL of different concentrations of R-, S-, and racemic-Bup was evaluated quantitatively by the fraction of times a pinprick failed to evoke a nocifensive motor response. R-Bup was more potent in the extent of block; however, S-Bup had a longer-lasting action at smaller doses. This significant difference was apparent when R-Bup and S-Bup were administered in equipotent doses of 0.06% and 0.075%, respectively. Co-injection of epinephrine (Epi) with these equipotent doses enhanced and prolonged the blocking effects of both Bup stereoisomers, although at dilutions of 1:100,000 to 1:1,000,000 Epi itself induced partial, transient analgesia. At 1:2,000,000 dilution, Epi alone had no analgesic effect; however, when co-injected with the shorter-acting R-Bup (0. 06%), Epi prolonged its blocking effect to equal the duration of block evoked by equipotent S-Bup (0.075%). We conclude R-Bup is more potent for cutaneous analgesia and that the longer duration of block by S-Bup probably originates from vasoconstrictor activity. IMPLICATIONS: Here we show that the more potent optical R-isomer of bupivacaine (Bup) can be used at a smaller dose (80%) than the S-isomer of Bup to give equal pain relief of a skin prick. Although the analgesia from R-Bup is briefer than that from equipotent S-Bup solutions, the durations become equal when a very dilute solution of the vasoconstrictor epinephrine is mixed with the R-isomer. The resulting vasoconstriction thus reduces vascular drug uptake and peak blood levels of systemic drug, reducing potential toxicity.

Near-terminus axonal structure and function following rat sciatic nerve regeneration through a collagen-GAG matrix in a ten-millimeter gap.

The objectives of this study were to evaluate the regenerated axon structure at near-terminal locations in the peroneal and tibial branches 1 year following implantation of several tubular devices in a 10-mm gap in the adult rat sciatic nerve and to determine the extent of recovery of selected sensory and motor functions. The devices were collagen and silicone tubes implanted alone or filled with a porous collagen-glycosaminoglycan matrix. Intact contralateral nerves and autografts were used as controls. Nerves were retrieved at 30 and 60 weeks postoperatively for histological evaluation of the number and diameter of regenerated axons proximal and distal to the gap and in the tibial and peroneal nerve branches, near the termination point. Several functional evaluation methods were employed: gait analysis, pinch test, muscle circumference, and response to electrical stimulation. A notable finding was that the matrix-filled collagen tube group had a significantly greater number of large-diameter myelinated axons (> or =6 microm in diameter) in the distal nerve branches than any other group, including the autograft group. These results were consistent with previously reported electrophysiological measurements that showed that the action potential amplitude for the A fibers in the matrix-filled collagen tube group was greater than for the autograft control group. Functional testing revealed the existence of both sensory and motor recovery following peripheral nerve regeneration through all devices; however, the tests employed in this study did not show differences among the groups with regeneration. Electrical stimulation in vivo showed that threshold parameters to elicit muscle twitch were the same for reinnervating and control nerves. The investigation is of importance in showing for the first time the superiority of a specific fully resorbable off-the-shelf device over an autograft for bridging gaps in peripheral nerve, with respect to the near-terminus axonal structure.

The local anesthetic properties and toxicity of saxitonin homologues for rat sciatic nerve block in vivo.

BACKGROUND AND OBJECTIVES: Saxitoxin and its homologues are naturally occurring compounds that block the sodium channel with high potency. They have the potential for providing prolonged duration local anesthesia when coinjected with vasoconstrictors or conventional local anesthetics and are devoid of local neurotoxicity. Here, we compare sciatic nerve block with saxitoxin to those with neosaxitoxin, decarbamoyl saxitoxin, and tetrodotoxin (TTX), in a search for even safer compounds. METHODS: Rats received percutaneous sciatic nerve block with toxins. The compounds were compared in terms of lethality, onset and duration of action for thermal analgesia (hot-plate testing), and motor block (weight-bearing). Data were expressed as medians with 25th and 75th percentiles, and median effective concentrations were determined. RESULTS: The median concentrations at which analgesia of 60 minutes duration was achieved were neosaxitoxin, 34+/-2 micromol/L; saxitoxin, 58+/-3 micromol/L; TTX, 92+/-5 micromol/L; and decarbamoyl saxitoxin, 268+/-8 micromol/L. Similar trends were observed for other measures of effectiveness (block duration of 90 minutes, maximal block), and for lethality so that the therapeutic indices were similar. No toxin had a marked predominance of sensory or motor block. The potency of TTX was intermediate between those of the saxitoxins, and its therapeutic index was slightly better. No difference was observed in time to onset of nerve blockade among the toxins. CONCLUSIONS: Substitutions on the saxitoxin nucleus result in large differences in incidence and duration of block, and toxicity. The therapeutic indices of the saxitoxins are similar; that of TTX is slightly better.

Beta-estradiol acutely potentiates the depression of cardiac excitability by lidocaine and bupivacaine.

Pregnancy is known to increase myocardial susceptibility to bupivacaine-induced cardiovascular collapse, and prolonged pretreatment of rabbits with high doses of progesterone potentiates bupivacaine's depression of the maximal rate of increase (Vmax) of the cardiac action potential. Short-term effects of progesterone are not detected in vitro, but other steroids elevated during pregnancy might be acutely active in this model. These experiments tested whether acute exposure to beta-estradiol potentiates local anesthetic/antiarrhythmic depression of Vmax and conduction velocity in rabbit cardiac tissue in vitro. Standard intracellular microelectrodes were used to measure electrophysiologic changes produced by beta-estradiol, local anesthetics, or both in dissected segments of heart containing the Purkinje fiber and ventricular muscle cells from ovariectomized rabbits. In tissues preincubated in beta-estradiol (3.3 nM), addition of bupivacaine (10.4 microM), or lidocaine (85.4 and 129 microM) decreased Vmax significantly more than in steroid-free Tyrode's (p<0.001). Alone, beta-estradiol had no effect on Vmax and depression of Vmax by the nonanesthetic Na+ channel blocker tetrodotoxin (TTX, 3 microM) was not potentiated by beta-estradiol. In preparations initially exposed to bupivacaine for 30 min, subsequent addition of beta-estradiol decreased Vmax further within 10 min (p<0.05). Bupivacaine's greater depression of Vmax at higher frequencies (3 Hz) was exaggerated by beta-estradiol. However, the rate-dependent slowing of conduction by bupivacaine was lessened or even reversed by beta-estradiol addition. Such rapid physiologic changes cannot be due to genomic actions by the hormone that take hours to manifest. Nor is the potentiation due to a general decrease in membrane excitability because the comparable inhibition by TTX is insensitive to estradiol. Because beta-estradiol potentiates the inhibition of myocardial excitability, but alleviates the slowing of impulse conduction between the Purkinje fiber and ventricular muscle produced by local anesthetics, the hormone must produce changes in more than one ionic conductance. Both pregnancy and conditions that abnormally alter levels of steroid hormones have ramifications for local anesthetic-induced cardiotoxicity and antiarrhythmic pharmacotherapeutics.