Shin Onizuka

Dural puncture with a 26-gauge spinal needle affects spread of epidural anesthesia

Combined spinal and epidural anesthesia may increase the risk of epidurally administered drugs spreading into the subarachnoid space through the dural hole.We studied the effect of dural puncture with a 26-gauge needle on the spread of analgesia induced by epidural injection of local anesthetics. Forty patients were randomly assigned to control and dural puncture groups. In the dural puncture group, the dura was punctured with a 26-gauge Whitacre spinal needle at L2-3 but no drug was injected. In both groups, an 18-gauge epidural catheter was inserted 4 cm cephalad into the epidural space at L2-3 and 15 mL of 2% mepivacaine without epinephrine was injected. Analgesia was assessed by pinprick at 5, 10, 15, and 20 min after injection and at the end of surgery. The caudal spread of analgesia was significantly greater in the dural puncture group than in the control group 15 and 20 min after injection (P < 0.01), but the cranial spread of analgesia was not different between the two groups. We conclude that dural puncture (without drugs) using a 26-gauge Whitacre spinal needle before epidural injection increases caudal spread of analgesia induced by epidural local anesthetics. (Anesth Analg 1996;82:1040-2)

Procaine and mepivacaine have less toxicity in vitro than other clinically used local anesthetics.

The neurotoxicity of local anesthetics can be demonstrated in vitro by the collapse of growth cones and neurites in cultured neurons. We compared the neurotoxicity of procaine, mepivacaine, ropivacaine, bupivacaine, lidocaine, tetracaine, and dibucaine by using cultured neurons from the freshwater snail Lymnaea stagnalis. A solution of local anesthetics was added to the culture dish to make final concentrations ranging from 1 × 10−6 to 2 × 10−2 M. Morphological changes in the growth cones and neurites were observed and graded 1 (moderate) or 2 (severe). The median concentrations yielding a score of 1 were 5 × 10−4 M for procaine, 5 × 10−4 M for mepivacaine, 2 × 10−4 M for ropivacaine, 2 × 10−4 M for bupivacaine, 1 × 10−4 M for lidocaine, 5 × 10−5 M for tetracaine, and 2 × 10−5 M for dibucaine. Statistically significant differences (P < 0.05) were observed between mepivacaine and ropivacaine, bupivacaine and lidocaine, lidocaine and tetracaine, and tetracaine and dibucaine. The order of neurotoxicity was procaine = mepivacaine < ropivacaine = bupivacaine < lidocaine < tetracaine < dibucaine. Although lidocaine is more toxic than bupivacaine and ropivacaine, mepivacaine, which has a similar pharmacological effect to lidocaine, has the least-adverse effects on cone growth among clinically used local anesthetics.

Up-regulation of NaV1.7 sodium channels expression by tumor necrosis factor-α in cultured bovine adrenal chromaffin cells and rat dorsal root ganglion neurons.

BACKGROUND:Tumor necrosis factor-&agr; (TNF-&agr;) is not only a key regulator of inflammatory response but also an important pain modulator. TNF-&agr; enhances both tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant Na+ channel currents in dorsal root ganglion (DRG) neurons. However, it remains unknown whether TNF-&agr; affects the function and expression of the TTX-S NaV1.7 Na+ channel, which plays crucial roles in pain generation. METHODS:We used cultured bovine adrenal chromaffin cells expressing the NaV1.7 Na+ channel isoform and compared them with cultured rat DRG neurons. The expression of TNF receptor 1 and 2 (TNFR1 and TNFR2) in adrenal chromaffin cells was studied by Semiquantitative reverse transcription-polymerase chain reaction. The effects of TNF-&agr; on the expression of NaV1.7 were examined with reverse transcription-polymerase chain reaction and Western blot analysis. Results were expressed as mean ± SEM. RESULTS:TNFR1 and TNFR2 were expressed in adrenal chromaffin cells, as well as reported in DRG neurons. TNF-&agr; up-regulated NaV1.7 mRNA by 132% ± 9% (N = 5, P = 0.004) in adrenal chromaffin cells, as well as 117% ± 2% (N = 5, P < 0.0001) in DRG neurons. Western blot analysis showed that TNF-&agr; increased NaV1.7 protein up to 166% ± 24% (N = 5, corrected P < 0.0001) in adrenal chromaffin cells, concentration- and time-dependently. CONCLUSIONS:TNF-&agr; up-regulated NaV1.7 mRNA in both adrenal chromaffin cells and DRG neurons. In addition, TNF-&agr; up-regulated the protein expression of the TTX-S NaV1.7 channel in adrenal chromaffin cells. Our findings may contribute to understanding the peripheral nociceptive mechanism of TNF-&agr;.

Capsaicin indirectly suppresses voltage-gated Na+ currents through TRPV1 in rat dorsal root ganglion neurons.

BACKGROUND:Capsaicin is used to treat a variety of types of chronic pain, including arthritis and trigeminal neuralgia. Although the cellular effects of capsaicin have been widely studied, little is known about the effects of capsaicin on intracellular sodium ([Na+]i) concentrations and voltage-gated Na+ currents (INa+) in nociceptive afferent neurons. Therefore, in this study we sought to characterize the effect of capsaicin on tetrodotoxin-sensitive (TTX-s) and resistant (TTX-r) INa+. METHODS:The effects of capsaicin on INa+ in rat dorsal root ganglion neurons were studied for both TTX-s and TTX-r components using whole-cell patch-clamp techniques and intracellular sodium imaging. RESULTS:In both TTX-s and TTX-r INa+ of capsaicin-sensitive neurons, capsaicin (0.1 to 10 &mgr;M) reduced inward currents in a dose-dependent manner. Capsaicin induced a hyperpolarization shift in the steady-state inactivation curves. SB366791 (10 &mgr;M), a potent and selective transient receptor potential vanilloid member1 (TRPV1) antagonist, significantly attenuated the reduction in INa+. Capsaicin induced an increase in the [Na+]i, and SB366791 (10 &mgr;M) significantly reduced the [Na+]i increase. An increase in [Na+]i with gramicidin also dependently suppressed INa+ and induced a hyperpolarization shift in the steady-state inactivation curves by increasing the [Na+]i. CONCLUSION:The findings suggest that capsaicin decreases both TTX-s and TTX-r INa+ as a result of an increase in [Na+]i through TRPV1.

Sevoflurane blocks cholinergic synaptic transmission postsynaptically but does not affect short-term potentiation.

Background: As compared with their effects on both inhibitory and excitatory synapses, little is known about the mechanisms by which general anesthetics affect synaptic plasticity that forms the basis for learning and memory at the cellular level. To test whether clinically relevant concentrations of sevoflurane affect short-term potentiation involving cholinergic synaptic transmission, the soma–soma synapses between identified, postsynaptic neurons were used. Methods: Uniquely identifiable neurons visceral dorsal 4 (presynaptic) and left pedal dorsal 1 (postsynaptic) of the mollusk Lymnaea stagnalis were isolated from the intact ganglion and paired overnight in a soma–soma configuration. Simultaneous intracellular recordings coupled with fluorescent imaging of the FM1-43 dye were made in either the absence or the presence of sevoflurane. Results: Cholinergic synapses, similar to those observed in vivo, developed between the neurons, and the synaptic transmission exhibited classic short-term, posttetanic potentiation. Action potential–induced (visceral dorsal 4), 1:1 excitatory postsynaptic potentials were reversibly and significantly suppressed by sevoflurane in a concentration-dependent manner. Fluorescent imaging with the dye FM1-43 revealed that sevoflurane did not affect presynaptic exocytosis or endocytosis; instead, postsynaptic nicotinic acetylcholine receptors were blocked in a concentration-dependent manner. To test the hypothesis that sevoflurane affects short-term potentiation, a posttetanic potentiation paradigm was used, and synaptic transmission was examined in either the presence or the absence of sevoflurane. Although 1.5% sevoflurane significantly reduced synaptic transmission between the paired cells, it did not affect the formation or retention of posttetanic potentiation at this synapse. Conclusions: This study demonstrates that sevoflurane blocks cholinergic synaptic transmission postsynaptically but does not affect short-term synaptic plasticity at the visceral dorsal 4–left pedal dorsal 1 synapse.

Long-term Exposure to Local but Not Inhalation Anesthetics Affects Neurite Regeneration and Synapse Formation between Identified Lymnaea Neurons

Background:General and local anesthetics are used in various combinations during surgical procedures to repair damaged tissues and organs, which in almost all instances involve nervous system functions. Because synaptic transmission recovers rapidly from various inhalation anesthetics, it is generally assumed that their effects on nerve regeneration and synapse formation that precede injury or surgery may not be as detrimental as that of their local counterparts. However, a direct comparison of most commonly used inhalation (sevoflurane, isoflurane) and local anesthetics (lidocaine, bupivacaine), vis-à-vis their effects on synapse transmission, neurite regeneration, and synapse formation has not yet been performed. Methods:In this study, using cell culture, electrophysiologic and imaging techniques on unequivocally identified presynaptic and postsynaptic neurons from the mollusc Lymnaea, the authors provided a comparative account of the effects of both general and local anesthetics on synaptic transmission, nerve regeneration, and synapse formation between cultured neurons. Results:The data show that clinically used concentrations of both inhalation and local anesthetics affect synaptic transmission in a concentration-dependent and reversal manner. The authors provided the first direct evidence that long-term overnight treatment of cultured neurons with sevoflurane and isoflurane does not affect neurite regeneration, whereas both lidocaine and bupivacaine suppress neurite outgrowth completely. The soma–soma synapse model was then used to compare the effects of both types of agents on synapse formation. The authors found that local but not inhalation anesthetics drastically reduced the incidence of synapse formation. The local anesthetic–induced prevention of synapse formation most likely involved the failure of presynaptic machinery, which otherwise developed normally in the presence of both sevoflurane and isoflurane. Conclusion:This study thus provides the first comparative, albeit preclinical, account of the effects of both general and local anesthetics on synaptic transmission, nerve regeneration, and synapse formation and demonstrates that clinically used lidocaine and bupivacaine have drastic long-term effects on neurite regeneration and synapse formation as compared with sevoflurane and isoflurane.

Lidocaine increases intracellular sodium concentration through voltage-dependent sodium channels in an identified lymnaea neuron.

Background:The local anesthetic lidocaine affects neuronal excitability in the central nervous system; however, the mechanisms of such action remain unclear. The intracellular sodium concentration ([Na+]i) and sodium currents (INa) are related to membrane potential and excitability. Using an identifiable respiratory pacemaker neuron from Lymnaea stagnalis, the authors sought to determine whether lidocaine changes [Na+]i and membrane potential and whether INa is related to these changes. Methods:Intracellular recording and sodium imaging were used simultaneously to measure membrane potentials and [Na+]i, respectively. Measurements for [Na+]i were made in normal, high-Na+, and Na+-free salines, with membrane hyperpolarization, and with tetrodotoxin pretreatment trials. Furthermore, changes of INa were measured by whole cell patch clamp configuration. Results:Lidocaine increased [Na+]i in a dose-dependent manner concurrent with a depolarization of the membrane potential. In the presence of high-Na+ saline, [Na+]i increased and the membrane potential was depolarized; the addition of lidocaine further increased [Na+]i, and the membrane potential was further depolarized. In Na+-free saline or in the presence of tetrodotoxin, lidocaine did not change [Na+]i. Similarly, hyperpolarization of the membrane by current injections also prevented the lidocaine-induced increase of [Na+]i. In the patch clamp configuration, membrane depolarization by lidocaine led to an inward sodium influx. A persistent reduction in membrane potential, resulting from lidocaine, brings the cell within the window current of INa where sodium channel activation occurs. Conclusion:Lidocaine increases intracellular sodium concentration and promotes excitation through voltage-dependent sodium channels by altering membrane potential in the respiratory pacemaker neuron.

Local Anesthetics with High Lipophilicity are Toxic, While Local Anesthetics with Low pka Induce More Apoptosis in Human Leukemia Cells

Many studies have indicated that local anesthetics are cytotoxic and can induce apoptosis; however, the types of local anesthetics and the induction rates of apoptosis remain unclear. The aim of this study was to clarify the local anesthetics that induce apoptosis or necrosis and their induction-related factors. Methods: Lidocaine, mepivacaine, bupivacaine, ropivacaine, tetracaine, dibucaine, procaine, and QX-314 were evaluated for apoptosis and necrosis in HL-60 human leukemia cell lines. Apoptosis and necrosis were analyzed by double-staining assay with propidium iodide (PI) and annexin-V and were measured by flow cytometry (FACS). DNA fragmentation was used for the analysis of apoptosis. Results: In the double-staining assay by flow cytometry, drugs with high lipophilicity were most cytotoxic. The comparative LD50 values were dibucaine > tetracaine > bupivacaine > ropivacaine > mepivacaine > lidocaine > procaine > QX-314. The LD50 were correlated with lipophilicity (logP). The comparative maximum rates of annexin-positive and PInegative apoptotic cells were lidocaine > mepivacaine > ropivacaine > bupivacaine > procaine > tetracaine > dibucaine > QX314 and were correlated with pKa. Lidocaine and mepivacaine significantly induced DNA fragmentation. DNA fragmentation was also correlated with pKa. Conclusion: The results indicate that local anesthetics with high lipophilicity are highly toxic and induce mainly necrosis, while local anesthetics with low pKa induce more apoptosis.

Lidocaine treatment during synapse reformation periods permanently inhibits NGF-induced excitation in an identified reconstructed synapse of Lymnaea stagnalis

PurposeNerve growth factor (NGF) has been reported to affect synaptic transmission and cause neuropathic pain. In contrast, lidocaine has been used to reduce neuropathic pain; however, the effect of NGF and lidocaine on spontaneous transmitter release and synapse excitation has not been fully defined. Therefore, the effect of NGF and lidocaine on nerve regeneration, synapse reformation, and subsequent spontaneous transmitter release was investigated. We used Lymnaea stagnalis soma–soma-identified synaptic reconstruction to demonstrate that a transient increase in both frequency and amplitude of spontaneous events of miniature endplate potentials (MEPPs) occurs following NGF treatment and a short burst of action potentials in the presynaptic cell; in addition, the effect of lidocaine on NGF-induced synapse reformation was investigated.MethodsUsing a cell culture and electrophysiological and FM-143 imaging techniques for exocytosis on unequivocally identified presynaptic visceral dorsal 4 (VD4) and postsynaptic somata left pedal (LPeE) neurons from the mollusc Lymnaea stagnalis, the effects of NGF and lidocaine on nerve regeneration, synapse reformation, and its electrophysiological spontaneous synaptic transmission between cultured neurons were described.ResultsNGF increased axonal growth, frequency, and amplitudes of MEPPs. Lidocaine exposure during synapse reformation periods was drastically and permanently reduced axonal growth and the incidence of synapse excitation by NGF.ConclusionNGF increased amplitudes and frequencies of MEPPs and induced synaptic excitation by increasing axonal growth and exocytosis. Lidocaine exposure during synapse reformation periods permanently suppressed NGF-induced excitation by suppressing axonal growth and exocytosis of presynaptic neurons in the identified reconstructed synapse of L. stagnalis.

Increase in intracellular Ca2+ concentration is not the only cause of lidocaine-induced cell damage in the cultured neurons of Lymnaea stagnalis

PurposeTo determine whether the increase in intracellular Ca2+ concentration induced by lidocaine produces neurotoxicity, we compared morphological changes and Ca2+ concentrations, using fura-2 imaging, in the cultured neurons of Lymnaea stagnalis.MethodsWe used BAPTA-AM, a Ca2+ chelator, to prevent the increase in the intracellular Ca2+ concentration, and Calcimycin A23187, a Ca2+ ionophore, to identify the relationship between increased intracellular Ca2+ concentrations and neuronal damage without lidocaine. Morphological changes were confirmed using trypan blue to stain the cells.ResultsIncreasing the dose of lidocaine increased the intracellular Ca2+ concentration; however, there was no morphological damage to the cells in lidocaine at 3 × 10−3 M. Lidocaine at 3 × 10−2 M increased the intracellular Ca2+ concentration in both saline (from 238 ± 63 to 1038 ± 156 nM) and Ca2+-free medium (from 211 ± 97 to 1046 ± 169 nM) and produced morphological damage and shrinkage, with the formation of a rugged surface. With the addition of BAPTA-AM, lidocaine at 3 × 10−2 M moderately increased the intracellular Ca2+ concentration (from 150 ± 97 to 428 ± 246 nM) and produced morphological damage. These morphologically changed cells were stained dark blue with trypan blue dye. The Ca2+ ionophore increased the intracellular Ca2+ concentration (from 277 ± 191 to 1323 ± 67 nM) and decreased it to 186 ± 109 nM at 60 min. Morphological damage was not observed during the 60 min, but became apparent a few hours later.ConclusionThese results indicated that the increase in intracellular Ca2+ concentration is not the only cause of lidocaine-induced cell damage.