Gary R. Strichartz

Molecular mechanisms of local anesthesia: a review

Impulse block by LA occurs through the inhibition of voltage-gated Na+ channels. Both protonated and neutral LAs can inhibit Na+ channels though interference with the conformational changes that underly the activation process (the sequence of events that occurs as channels progress from the closed resting state to the open conducting state). The occlusion of open channels contributes little to the overall inhibition. Local anesthetic inhibition of Na+ currents increases with repetitive depolarizations in a process called phasic block. Phasic block represents increased LA binding, either because more channels become accessible during depolarization or because the channel conformations favored by depolarization bind LA with higher affinity. The details of phasic block are dependent on LA chemistry: certain LAs bind and dissociate quite rapidly, others act more slowly; some LAs interact effectively with closed states that occur intermediately between resting and open states, others favor the open channel, and still others have a higher affinity for inactivated states. Channel activation accelerates LA binding, and LAs may bind more tightly to activated and inactivated than to resting channels. In this regard, both the modulated receptor and the guarded receptor hypotheses are valid. In binding to activated and inactivated channels, LAs prevent the conformational changes of activation and antagonize the binding of activator agents that poise channels in activated, open states. These reciprocal actions are one aspect of the concerted conformational rearrangements that occur throughout Na+ channels during gating. The LA binding site may exist in the channels pore, at the membrane-protein interface, or within the protein subunits of the channel. Judging from its susceptibility to intracellular proteases and its accessibility to LAs with limited membrane permeability (i.e., quaternary LAs in the cytoplasm), the site lies nearer to the cytoplasmic than the external surface of the membrane. Nevertheless, protons in the external medium influence the dissociation of LA from the closed channel. Binding of LAs at the inhibitory site is weak and loose. If one accounts for the membrane-concentrating effects of LA hydrophobicity that are expressed as membrane: buffer partition coefficients equal to 10(2)-10(4), then the apparent LA affinities are low. The equilibrium dissociation constants calculated on the basis of free drug in the membrane are 1-10 mM, with a correspondingly weak binding to the inhibitory LA site. The stereospecificity of LA action is also relatively nonselective, suggesting a loose fit between ligand and binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

MAP kinase and pain

Mitogen-activated protein kinases (MAPKs) are important for intracellular signal transduction and play critical roles in regulating neural plasticity and inflammatory responses. The MAPK family consists of three major members: extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK), which represent three separate signaling pathways. Accumulating evidence shows that all three MAPK pathways contribute to pain sensitization after tissue and nerve injury via distinct molecular and cellular mechanisms. Activation (phosphorylation) of MAPKs under different persistent pain conditions results in the induction and maintenance of pain hypersensitivity via non-transcriptional and transcriptional regulation. In particular, ERK activation in spinal cord dorsal horn neurons by nociceptive activity, via multiple neurotransmitter receptors, and using different second messenger pathways plays a critical role in central sensitization by regulating the activity of glutamate receptors and potassium channels and inducing gene transcription. ERK activation in amygdala neurons is also required for inflammatory pain sensitization. After nerve injury, ERK, p38, and JNK are differentially activated in spinal glial cells (microglia vs astrocytes), leading to the synthesis of proinflammatory/pronociceptive mediators, thereby enhancing and prolonging pain. Inhibition of all three MAPK pathways has been shown to attenuate inflammatory and neuropathic pain in different animal models. Development of specific inhibitors for MAPK pathways to target neurons and glial cells may lead to new therapies for pain management. Although it is well documented that MAPK pathways can increase pain sensitivity via peripheral mechanisms, this review will focus on central mechanisms of MAPKs, especially ERK.

A Peptide c-Jun N-Terminal Kinase (JNK) Inhibitor Blocks Mechanical Allodynia after Spinal Nerve Ligation: Respective Roles of JNK Activation in Primary Sensory Neurons and Spinal Astrocytes for Neuropathic Pain Development and Maintenance

Optimal management of neuropathic pain is a major clinical challenge. We investigated the involvement of c-Jun N-terminal kinase (JNK) in neuropathic pain produced by spinal nerve ligation (SNL) (L5). SNL induced a slow (>3 d) and persistent (>21 d) activation of JNK, in particular JNK1, in GFAP-expressing astrocytes in the spinal cord. In contrast, p38 mitogen-activated protein kinase activation was found in spinal microglia after SNL, which had fallen to near basal level by 21 d. Intrathecal infusion of a JNK peptide inhibitor, D-JNKI-1, did not affect normal pain responses but potently prevented and reversed SNL-induced mechanical allodynia, a major symptom of neuropathic pain. Intrathecal D-JNKI-1 also suppressed SNL-induced phosphorylation of the JNK substrate, c-Jun, in spinal astrocytes. However, SNL-induced upregulation of GFAP was not attenuated by spinal D-JNKI-1 infusion. Furthermore, SNL induced a rapid (<12 h) but transient activation of JNK in the L5 (injured) but not L4 (intact) DRG. JNK activation in the DRG was mainly found in small-sized C-fiber neurons. Infusion of D-JNKI-1 into the L5 DRG prevented but did not reverse SNL-induced mechanical allodynia. Finally, intrathecal administration of an astroglial toxin, l-α-aminoadipate, reversed mechanical allodynia. Our data suggest that JNK activation in the DRG and spinal cord play distinct roles in regulating the development and maintenance of neuropathic pain, respectively, and that spinal astrocytes contribute importantly to the persistence of mechanical allodynia. Targeting the JNK pathway in spinal astroglia may present a new and efficient way to treat neuropathic pain symptoms.

Cell Signaling and the Genesis of Neuropathic Pain

Damage to the nervous system can cause neuropathic pain, which is in general poorly treated and involves mechanisms that are incompletely known. Currently available animal models for neuropathic pain mainly involve partial injury of peripheral nerves. Multiple inflammatory mediators released from damaged tissue not only acutely excite primary sensory neurons in the peripheral nervous system, producing ectopic discharge, but also lead to a sustained increase in their excitability. Hyperexcitability also develops in the central nervous system (for instance, in dorsal horn neurons), and both peripheral and spinal elements contribute to neuropathic pain, so that spontaneous pain may occur or normally innocuous stimuli may produce pain. Inflammatory mediators and aberrant neuronal activity activate several signaling pathways [including protein kinases A and C, calcium/calmodulin-dependent protein kinase, and mitogen-activated protein kinases (MAPKs)] in primary sensory and dorsal horn neurons that mediate the induction and maintenance of neuropathic pain through both posttranslational and transcriptional mechanisms. In particular, peripheral nerve lesions result in activation of MAPKs (p38, extracellular signal–regulated kinase, and c-Jun N-terminal kinase) in microglia or astrocytes in the spinal cord, or both, leading to the production of inflammatory mediators that sensitize dorsal horn neurons. Activity of dorsal horn neurons, in turn, enhances activation of spinal glia. This neuron-glia interaction involves positive feedback mechanisms and is likely to enhance and prolong neuropathic pain even in the absence of ongoing peripheral external stimulation or injury. The goal of this review is to present evidence for signaling cascades in these cell types that not only will deepen our understanding of the genesis of neuropathic pain but also may help to identify new targets for pharmacological intervention. Nerve injury not only leads to immediate sensations of pain, which serves an adaptive function, but can also lead to chronic pain, which is pathological. Although the mechanisms underlying the genesis and maintenance of neuropathic pain—the chronic pain that often, although in humans not always, results from nerve injury—remain incompletely understood, they are known to involve signaling pathways initiated both by inflammatory substances released from the damaged tissues and by aberrant firing of the damaged neurons. These signaling pathways lead to posttranslational modifications of existing proteins that appear to be involved in the genesis of neuropathic pain, as well as to changes in transcriptional activity that appear to be involved in its maintenance. This STKE Review explores the peripheral and central mechanisms underlying the initiation and maintenance of neuropathic pain and discusses the interactions between neurons and glia that are likely to prolong and enhance the pain that results from nerve injury. A deeper understanding of the mechanisms that contribute to neuropathic pain may be expected to lead to more effective methods of treating this chronic and disabling condition.

Irreversible Conduction Block in Isolated Nerve by High Concentrations of Local Anesthetics

Background:Delivery of large doses of local anesthetics for spinal anesthesia by repeated injections or continuous infusion could expose the cauda equina to concentrations of drug that may be neurotoxic per se. We studied this possible neurotoxic effect by assessing recovery from conduction blockade of de-sheathed peripheral nerves after exposure to some of the local anesthetic solutions commonly used for spinal anesthesia. Methods:The reversibility of conduction blockade was studied in desheathed bullfrog sciatic nerves, using the sucrose-gap method for recording compound action potentials, before and during exposure to local anesthetics and during drug washout. The nerves were exposed for 15 min to 5% or 1.5% lidocaine, with or without 7.5% dextrose; 0.5% tetracaine; or 0.75% bupivacaine (the latter two without dextrose). Some nerves were also bathed in 7.5% dextrose (without local anesthetic) or in 0.06% tetracaine, which in this preparation is equipotent to 5% lidocaine. After 15 min in the drug, the nerves were washed for 2–3 h and soaked in Ringers solution overnight. Nerves exposed only to Ringers solution served as controls. We also studied neuronal uptake and washout of radio-labeled lidocaine. Results:Exposure of nerves to 5% lidocaine, with or without 7.5% dextrose, or to 0.5% tetracaine resulted in irreversible total conduction blockade, whereas 1.5% lidocaine or 0.75% bupivacaine caused 25–50% residual block after the 2–3-h wash. Nerves exposed to Ringers solution, 7.5% dextrose or 0.06% tetracaine had 0–10% residual block after 2–3 h wash. The action potential of all nerves declined after overnight soak to between 30–60% of the control value, except for those nerves exposed to 5% lidocaine or 0.5% tetracaine, which showed no activity. Exposure to 5% lidocaine for periods of only 4–5 min produced total, irreversible loss of conduction. The uptake by and washout of radiolabeled lidocaine from the nerves indicate that the maximum amount of residual drug after 2–4 min of exposure to 5% lidocaine and a 3-h wash should cause at most only 50% conduction block. Conclusions:Solutions of 5% lidocaine and 0.5% tetracaine that have been associated with clinical cases of cauda equina syndrome after continuous spinal anesthesia caused irreversible conduction block in desheathed amphibian nerve. Whether these in vitro actions also occur in mammalian nerves in vivo is an important, clinically relevant question now under investigation in our laboratory.

Lipid infusion accelerates removal of bupivacaine and recovery from bupivacaine toxicity in the isolated rat heart.

Background and Objectives: Infusion of a lipid emulsion has been advocated for treatment of severe bupivacaine cardiac toxicity. The mechanism of lipid rescue is unknown. These studies address the possibility that lipid infusion reduces cardiac bupivacaine content in the context of cardiac toxicity. Methods: We compared the effects of a 1% lipid emulsion with standard Krebs buffer after inducing asystole in isolated rat heart with 500 μmol/L bupivacaine. We compared times to first heart beat and recovery of 90% of baseline rate pressure product (RPP = heart rate × [left ventricular systolic pressure − left ventricular diastolic pressure]) between controls and hearts receiving 1% lipid immediately after bupivacaine. We also used minibiopsies to compare control bupivacaine tissue content with hearts getting lipid immediately after an infusion of radiolabeled bupivacaine. We then compared bupivacaine efflux from hearts with and without lipid infusion started 75 seconds after radiolabeled bupivacaine was administered. Results: Infusion of lipid resulted in more rapid return of spontaneous contractions and full recovery of cardiac function. Average (± SEM) times to first beat and to 90% recovery of rate pressure product were 44.6 ± 3.5 versus 63.8 ± 4.3 seconds (P < .01) and 124.7 ± 12.4 versus 219.8 ± 25.6 seconds (P < .01) for lipid and controls, respectively. Lipid treatment resulted in more rapid loss of bupivacaine from heart tissue (P < .0016). Late lipid infusion, 75 seconds after bupivacaine infusion ended, increased the release of bupivacaine measured in effluent for the first 15-second interval compared with controls (183 vs. 121 nmol, n = 5 for both groups, P < .008). Conclusions: Lipid emulsion speeds loss of bupivacaine from cardiac tissue while accelerating recovery from bupivacaine-induced asystole. These findings are consistent with the hypothesis that bupivacaine partitions into the emulsion and supports the concept of a “lipid sink.” However, the data do not exclude other possible mechanisms of action.

Fundamental properties of local anesthetics. II. Measured octanol:buffer partition coefficients and pKa values of clinically used drugs.

Because local anesthetic molecules interact with ion channel proteins embedded in membranes to effect impulse blockade, and because their clinical potency often depends on both vascular absorption and distribution into the tissue surrounding the site of deposition, the ability to partition into these various compartments is an important determinant of local anesthetic action. Therefore, the hydrophobic nature of local anesthetics used clinically was characterized by the octanol:bufer partition coefficients of their charged (P+) and neutral (P0) species. This was accomplished by previously described optical methods in which direct spectrophotometric measurement of both the pH-dependent distribution coefficient (Q) and of the ionization permit calculation of the pKa and partition coefficients. The rates of alkaline hydrolysis of ester-linked molecules also were measured to assess potential interference of such hydrolysis with the physicochemical assays. Results indicate that the hydrophobicity of a local anesthetic is increased by manipulation of the molecular structure at three sites: (a) the aromatic ring; (b) the intermediate linking group; and (c) the tertiary amine. P0 for the agents studied was 103-105 times greater than P+. Although there is no systematic relationship between hydrophobicity and pKa, the latter is greater with ester-linked (pKa = 8.59−9.30) than with amide-linked (pKa = 7.92−8.21) local anesthetics. All of the charged species, with the exception of bupivacaine, selectively partition into the aqueous environment (P+ < 1.0). The temperature dependence of partitioning of the local anesthetics, measured at 25 and 36°C, indicates an entropy-driven hydrophobic uptake. Solutions bufered with bicarbonate and including 5% CO2 showed the same local anesthetic partitioning as that of CO2-free solutions, suggesting that potentiation of impulse blockade by CO2 is not due to increased membrane uptake. Correlations of physicochemical properties of local anesthetics with potencies on isolated nerve confirm that the more potent local anesthetics have greater octanol:buffer partition coefficients, and that the ester-linked local anesthetics are more potent than their amide-linked counterparts having the same hydrophobicities. The correlations of structure with potency also suggest that the extracellular protonated species may contribute to impulse blockade.

Collagen-GAG substrate enhances the quality of nerve regeneration through collagen tubes up to level of autograft.

Peripheral nerve regeneration was studied across a tubulated 10-mm gap in the rat sciatic nerve using histomorphometry and electrophysiological measurements of A-fiber, B-fiber, and C-fiber peaks of the evoked action potentials. Tubes fabricated from large-pore collagen (max. pore diameter, 22 nm), small-pore collagen (max. pore diameter, 4 nm), and silicone were implanted either saline-filled or filled with a highly porous, collagen-glycosaminoglycan (CG) matrix. The CG matrix was deliberately synthesized, based on a previous optimization study, to degrade with a half-life of about 6 weeks and to have a very high specific surface through a combination of high pore volume fraction (0.95) and relatively small average pore diameter (35 microm). Nerves regenerated through tubes fabricated from large-pore collagen and filled with the CG matrix had significantly more large-diameter axons, more total axons, and significantly higher A-fiber conduction velocities than any other tubulated group; and, although lower than normal, their histomorphometric and electrophysiological properties were statistically indistinguishable from those of the autograft control. Although the total number of myelinated axons in nerves regenerated by tubulation had reached a plateau by 30 weeks, the number of axons with diameter larger than 6 microm, which have been uniquely associated with the A-fiber peak of the action potential, continued to increase at substantial rates through the completion of the study (60 weeks). The kinetic data strongly suggest that a nerve trunk maturation process, not previously reported in studies of the tubulated 10-mm gap in the rat sciatic nerve, and consisting in increase of axonal tissue area with decrease in total tissue area, continues beyond 60 weeks after injury, resulting in a nerve trunk which increasingly approaches the structure of the normal control.

Endothelin-B receptor activation triggers an endogenous analgesic cascade at sites of peripheral injury

Endothelin-1 (ET-1) is a newly described pain mediator that is involved in the pathogenesis of pain states ranging from trauma to cancer. ET-1 is synthesized by keratinocytes in normal skin and is locally released after cutaneous injury. While it is able to trigger pain through its actions on endothelin-A (ETA) receptors of local nociceptors, it can coincidentally produce analgesia through endothelin-B (ETB) receptors. Here we map a new endogenous analgesic circuit, in which ETB receptor activation induces the release of β-endorphin from keratinocytes and the activation of G-protein-coupled inwardly rectifying potassium channels (GIRKs, also named Kir-3) linked to opioid receptors on nociceptors. These results indicate the existence of an intrinsic feedback mechanism to control peripheral pain in skin, and establish keratinocytes as an ETB receptor–operated opioid pool.

Emerging roles of resolvins in the resolution of inflammation and pain

Resolvins, including D and E series resolvins, are endogenous lipid mediators generated during the resolution phase of acute inflammation from the omega-3 polyunsaturated fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Resolvins have potent anti-inflammatory and pro-resolution actions in several animal models of inflammation. Recent findings also demonstrate that resolvin E1 and resolvin D1 can each potently dampen inflammatory and postoperative pain. This review focuses on the mechanisms by which resolvins act on their receptors in immune cells and neurons to normalize exaggerated pain via regulation of inflammatory mediators, transient receptor potential (TRP) ion channels, and spinal cord synaptic transmission. Resolvins may offer novel therapeutic approaches for preventing and treating pain conditions associated with inflammation.