Nerve Cell Model Research Articles

Local anesthetic failure associated with inflammation: verification of the acidosis mechanism and the hypothetic participation of inflammatory peroxynitrite

The presence of inflammation decreases local anesthetic efficacy, especially in dental anesthesia. Although inflammatory acidosis is most frequently cited as the cause of such clinical phenomena, this has not been experimentally proved. We verified the acidosis mechanism by studying the drug and membrane lipid interaction under acidic conditions together with proposing an alternative hypothesis. Liposomes and nerve cell model membranes consisting of phospholipids and cholesterol were treated at different pH with lidocaine, prilocaine and bupivacaine (0.05%-0.2%, w/v). Their membrane-interactive potencies were compared by the induced-changes in membrane fluidity. Local anesthetics fluidized phosphatidylcholine membranes with the potency being significantly lower at pH 6.4 than at pH 7.4 (p < 0.01), supporting the acidosis theory. However, they greatly fluidized nerve cell model membranes even at pH 6.4 corresponding to inflamed tissues, challenging the conventional mechanism. Local anesthetics acted on phosphatidylserine liposomes, as well as nerve cell model membranes, at pH 6.4 with almost the same potency as that at pH 7.4, but not on phosphatidylcholine, phosphatidylethanolamine and sphingomyelin liposomes. Since the positively charged anesthetic molecules are able to interact with nerve cell membranes by ion-paring with anionic components like phosphatidylserine, tissue acidosis is not essentially responsible for the local anesthetic failure associated with inflammation. The effects of local anesthetics on nerve cell model membranes were inhibited by treating with peroxynitrite (50 μM), suggesting that inflammatory cells producing peroxynitrite may affect local anesthesia.

Peroxynitrite affects lidocaine by acting on membrane-constituting lipids

Inflammation frequently decreases local anesthetic effects, especially in dental anesthesia in patients with pulpitis and periodontitis. The pharmacokinetics and the mode of action of local anesthetics are closely associated with the hydrophobic interactions between these drugs and lipid bilayers that change the membrane physicochemical property, fluidity. A lipid oxidant, peroxynitrite, is produced by inflammatory cells, and it may act on nerve cell membranes and affect anesthetic efficacy. With respect to this speculated action, we addressed whether peroxynitrite acted on membrane-constituting lipids to decrease the membrane interactivity of lidocaine. Membrane fluidity changes were determined by measuring the fluorescence polarization of liposomes prepared with different phospholipids. Peroxynitrite (0.1-50 microM) rigidified nerve-cell model membranes consisting of unsaturated phospholipids, as well as liposomal membranes consisting of 1,2-dioleoylphosphatidylcholine and 1-stearoyl-2-arachidonylphosphatidylcholine, but peroxynitrite did not rigidify 1, 2-dipalmitoylphosphatidylcholine liposomal membranes. The pretreatment of nerve-cell model membranes with peroxynitrite (0.1-50 microM) decreased the membrane-fluidizing effects of lidocaine (5.0 mg x ml(-1)) to 63%-86% of the control (not treated with peroxynitrite) depending on the peroxynitrite concentration. As one of the mechanisms of the local anesthetic failure associated with inflammation, inflammatory peroxynitrite may affect local anesthesia by acting on membrane-constituting unsaturated phospholipids.

Membrane interactivity of charged local anesthetic derivative and stereoselectivity in membrane interaction of local anesthetic enantiomers

With respect to the membrane lipid theory as a molecular mechanism for local anesthetics, two critical subjects, the negligible effects of charged drugs when applied extracellularly and the stereoselective effects of enantiomers, were verified by paying particular attention to membrane components, phospholipids with the anionic property, and cholesterol with several chiral carbons. The membrane interactivities of structurally-different anesthetics were determined by their induced fluidity changes of liposomal membranes. Lidocaine (3.0 μmol/mL) fluidized phosphatidylcholine membranes, but not its quaternary derivative QX-314 (3.0 μmol/mL). Similarly to the mother molecule lidocaine, however, QX-314 fluidized phosphatidylserine-containing nerve cell model membranes and acidic phospholipids-constituting membranes depending on the acidity of membrane lipids. Positively charged local anesthetics are able to act on lipid bilayers by ion-pairing with anionic (acidic) phospholipids. Bupivacaine (0.75 mol/mL) and ropivacaine (0.75 and 1.0 μmol/mL) fluidized nerve cell model membranes with the potency being S(-)-enantiomer < racemate < R(+)-enantiomer (P < 0.01, vs antipode and racemate) and cardiac cell model membranes with the potency being S(-)-ropivacaine < S(-)-bupivacaine < R(+)-bupivacaine (P < 0.01). However, their membrane effects were not different when removing cholesterol from the model membranes. Stereoselectivity is producible by cholesterol which increases the chirality of lipid bilayers and enables to discriminate anesthetic enantiomers. The membrane lipid interaction should be reevaluated as the mode of action of local anesthetics.

Stereospecific Interaction of Bupivacaine Enantiomers with Lipid Membranes

S(-)-Bupivacaine has the pharmacotoxicological advantage over its antipode and racemate. The interaction with lipid membranes was compared between S(-)-, R(+)- and racemic bupivacaine. The bupivacaine-induced changes in membrane property were determined by turbidity and fluorescence polarization measurements of membrane preparations to which bupivacaine stereoisomers of 1.0-5.0 mmol/L were applied. Liposomal membranes were made of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine without or with cholesterol (5 to 15 mol%), and nerve cell model membranes of 55 mol% different phospholipids and 45 mol% cholesterol. The purity and hydrophobic interaction of bupivacaine were analyzed by reversed-phase high-performance liquid chromatography. Both S(-)- and R(+)-bupivacaine were not different in lowering the phase transition temperature of membrane 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. S(-)-, R(+)- and racemic bupivacaine disordered 100 mol% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposomal membranes, although the potency was indistinguishable between stereoisomers. By adding cholesterol to membranes, however, the membrane-disordering effects showed stereostructure-specificity that was enhanced with increasing the cholesterol content (0 to 15 mol%). The enantio-differentiating effects resulted from neither impurities in enantiomers nor hydrophobic interaction with phosphatidylcholine acyl chains. Bupivacaine disordered nerve cell model membranes with the potency being S(-)-enantiomer < racemate < R(+)-enantiomer, which resembled their relative stereopotency in nerve and cardiac channel inhibition. Membrane-disordering stereospecificity disappeared in the membranes without containing cholesterol. Bupivacaine stereostructure-specifically interacts with membranes containing cholesterol, which is consistent with the clinical features of S(-)-bupivacaine. Membrane cholesterol appears to increase the chirality of lipid bilayers and enable them to interact with S(-)-, racemic and R(+)-bupivacaine differently.

Assessment of multiple frequency ELF electric and magnetic field exposure

Electromagnetic fields both in daily life and at workplaces exhibit increasingly complex frequency spectra. Present spectral assessment rules proved to be too conservative for health risk assessment. This is because they are based on the assumption that cells would react like linear systems in terms of responding to a sum of frequencies by a sum of independent responses to each individual frequency. Based on numerical investigations with the Hodgkin–Huxley and the Frankenhaeuser–Huxley nerve cell models, it could be shown that accounting for the nonlinear behaviour of cellular excitation processes avoids considerable overestimation of simultaneous exposures to multiple frequency ELF electric and magnetic fields. Besides this, it could be shown that the role of phase relationships is less important than that assumed so far. The present assessment rules lead to non-compliances of marketed electric appliances. For general application, a nonlinear biology-based assessment (NBBA) rule has been proposed, validated and proven advantageous compared with ICNIRP's rule. While staying conservative it avoids unnecessary overestimation and demonstrates compliance even in cases of suspected non-conformities. It is up to responsible bodies to decide upon the adoption of this proposal and the potential need for implementing additional or reducing the already incorporated safety factors.

Interaction of local anaesthetics with lipid membranes under inflammatory acidic conditions

The clinical fact that local anaesthetics do not successfully work in the patients with inflammation has been generally interpreted on the basis of inflamed tissue acidification. In order to verify this hypothesis, the interaction of local anaesthetics with lipid membranes was studied by determining the drug-induced changes of membrane physicochemical property (membrane fluidity) at different pH covering inflammatory acidic conditions. At clinically relevant concentrations, lidocaine, procaine, prilocaine and bupivacaine fluidized 1,2-dipalmitoylphosphatidylcholine membranes with the potency decreased with lowering the pH from 7.9 to 5.9. When treated as the aqueous acidic solution (pH 4.0) similar to marketed injection solutions, lidocaine showed more pronounced pH dependence, so the reduction of its membrane-fluidizing effects at acidic pH theoretically correlated to that of its non-ionized membrane-interactive concentrations. Unlike phosphatidylcholine membranes, however, nerve cell model membranes consisting of different phospholipids and cholesterol were fluidized by lidocaine at pH 6.4-6.9 corresponding to the acidity of inflamed tissues. Cationic lidocaine was effective in fluidizing anionic phosphatidylserine and cardiolipin membranes at pH 6.4, but not zwitterionic phospholipid membranes, whereas it was ineffective on any membranes at pH 2.0 where membrane acidic phospholipids were not ionized. Local anaesthetics are considered to form the ion-pairs specifically with counter-ionic phospholipids and act on the membranes of nerve cells even under inflammatory acidic conditions. The drug and membrane interaction causable in inflamed tissue acidification does not support the conventional theory on the local anaesthetic failure associated with inflammation.

2603 血管・神経細胞微視モデルを用いた頭部損傷マルチスケール有限要素解析(OS26.並列・マルチスケール計算手法の開発と応用(1))

2603 血管・神経細胞微視モデルを用いた頭部損傷マルチスケール有限要素解析(OS26.並列・マルチスケール計算手法の開発と応用(1))

Development of a Multi-Scale Finite Element Analysis Code by Using a Micro Nerve Cell Model for Brain Injury Prediction

When the human head is loaded by the translational acceleration in the traffic crash accident, the human brain is damaged at the crash side and the counter side, such as the coup and the contre-coup brain injuries. The purpose of this study is to reveal a generation mechanism of the coup and contrecoup brain injuries and develop a prediction method of the brain injury at the nerve cell level by using the dynamic-explicit finite element method (FEM). We carried out a multi-scale crash simulation by using a macroscopic head model and a microscopic nerve cell model. We have investigated the hydrostatic pressure, the equivalent stress and strains distributions in the nerve cell during the head crash against the street plate. Numerical results show the availability of our multi-scale analysis to predict the brain injury at the macro and micro levels.

507 神経細胞微視モデルを用いた脳損傷有限要素解析(S10-2 衝突安全と生体工学(1),S10 衝突安全と生体工学)

When the human head is loaded by the translational acceleration in the traffic accident, the human brain is damaged at the crash side and the counter side (i.e., coup and contre-coup brain injuries). The purpose of this study is to investigate a generation mechanism of coup and contre-coup brain injury and to develop a prediction method of the brain injury at the nerve cell level by using the finite element method (FEM). We carried out a multi-scale crash simulation by using a macroscopic head model and a microscopic nerve cell model. We investigated the hydrostatic pressure distribution in nerve cell. The results show the availability of our multi-scale analysis.

Biphasic effects of oxethazaine, a topical anesthetic, on the intracellular Ca 2+ concentration of PC12 cells

There have been few reports on the mechanism(s) of action of oxethazaine (OXZ) despite its potent local anesthetic action. Generally, local anesthetics (LAs) not only inhibit Na + channels but also affect various membrane functions. In the present study, using PC12 cells as a nerve cell model, the effects of OXZ on intracellular Ca 2+ concentration ([Ca 2+] i) were examined in relation to cytotoxicity and dopamine release. [Ca 2+] i was determined by the quin2 method. In resting cells, (6–10)×10 −5 M OXZ produced lactate dehydrogenase leakage, which was Ca 2+-dependent, inhibited by metal Ca 2+ channel blockers, and preceded by a marked increase in [Ca 2+] i. Some other LAs showed no cytotoxicity at these concentrations. In K +-depolarized cells, however, lower concentrations of OXZ (10 −6–10 −7 M), that had no effect on resting [Ca 2+] i, inhibited both the dopamine release and the increase of [Ca 2+] i in parallel. The inhibitory potency against the [Ca 2+] i increase was in the order of nifedipine>OXZ∼verapamil>diltiazem, and OXZ acted additively on the Ca 2+ channel blockers. OXZ showed the least effect on K +-depolarization as determined by bisoxonol uptake. OXZ also inhibited the increase in [Ca 2+] i induced by S(−)-BAY K 8644, a Ca 2+ channel agonist. These observations suggested that low concentrations of OXZ interact with L-type Ca 2+ channels. The biphasic effects of OXZ on Ca 2+ movement may be due to a unique chemical structure, and may participate in and complicate the understanding of the potent pharmacological and toxicological actions of OXZ.

Nervous system-derived chondroitin sulfate proteoglycans regulate growth cone morphology and inhibit neurite outgrowth: a light, epifluorescence, and electron microscopy study.

Proteoglycans influence aging and plasticity in the nervous system. Particularly prominent are the chondroitin sulfate proteoglycans (CSPGs), which are generally inhibitory to neurite outgrowth. During development, CSPGs facilitate normal guidance, but following nervous system injury and in diseases of aging (e.g., Alzheimer's disease), they block successful regeneration, and are associated with axon devoid regions and degenerating nerve cells. Whereas previous studies used non-nervous system sources of CSPGs, this study analyzed the morphology and behavior of sensory (dorsal root ganglia) neurons, and a human nerve cell model (SH-SY5Y neuroblastoma cells) as they contacted nervous system-derived CSPGs, using a variety of microscopy techniques. The results of these qualitative analyses show that growth cones of both nerve cell types contact CSPGs via actin-based filopodia, sample the CSPGs repeatedly without collapse, and alter their trajectory to avoid nervous system-derived CSPGs. Turning and branching are correlated with increased filopodial sampling, and are common to both neurons and Schwann cells. We show that CSPG expression by rat CNS astrocytes in culture is correlated with sensory neuron avoidance. Further, we show for the first time the ultrastructure of sensory growth cones at a CSPG-laminin border and reveal details of growth cone and neurite organization at this choice point. This type of detailed analysis of the response of growth cones to nervous system-derived CSPGs may lead to an understanding of CSPG function following injury and in diseases of aging, where CSPGs are likely to contribute to aberrant neurite outgrowth, failed or reduced synaptic connectivity, and/or ineffective plasticity.

Regulation of antioxidant metabolism by translation initiation factor 2alpha.

Oxidative stress and highly specific decreases in glutathione (GSH) are associated with nerve cell death in Parkinson's disease. Using an experimental nerve cell model for oxidative stress and an expression cloning strategy, a gene involved in oxidative stress-induced programmed cell death was identified which both mediates the cell death program and regulates GSH levels. Two stress-resistant clones were isolated which contain antisense gene fragments of the translation initiation factor (eIF)2alpha and express a low amount of eIF2alpha. Sensitivity is restored when the clones are transfected with full-length eIF2alpha; transfection of wild-type cells with the truncated eIF2alpha gene confers resistance. The phosphorylation of eIF2alpha also results in resistance to oxidative stress. In wild-type cells, oxidative stress results in rapid GSH depletion, a large increase in peroxide levels, and an influx of Ca(2+). In contrast, the resistant clones maintain high GSH levels and show no elevation in peroxides or Ca(2+) when stressed, and the GSH synthetic enzyme gamma-glutamyl cysteine synthetase (gammaGCS) is elevated. The change in gammaGCS is regulated by a translational mechanism. Therefore, eIF2alpha is a critical regulatory factor in the response of nerve cells to oxidative stress and in the control of the major intracellular antioxidant, GSH, and may play a central role in the many neurodegenerative diseases associated with oxidative stress.

Ca2+ waves in PC12 neurites: a bidirectional, receptor-oriented form of Ca2+ signaling.

Spatial and temporal aspects of Ca2+ signaling were investigated in PC12 cells differentiated with nerve growth factor, the well known nerve cell model. Activation of receptors coupled to polyphosphoinositide hydrolysis gave rise in a high proportion of the cells to Ca2+ waves propagating non decrementally and at constant speed (2-4 microns/s at 18 degrees C and approximately 10-fold faster at 37 degrees C) along the neurites. These waves relied entirely on the release of Ca2+ from intracellular stores since they could be generated even when the cells were incubated in Ca(2+)-free medium. In contrast, when the cells were depolarized with high K+ in Ca(2+)-containing medium, increases of cytosolic Ca2+ occurred in the neurites but failed to evolve into waves. Depending on the receptor agonist employed (bradykinin and carbachol versus ATP) the orientation of the waves could be opposite, from the neurite tip to the cell body or vice versa, suggesting different and specific distribution of the responsible surface receptors. Cytosolic Ca2+ imaging results, together with studies of inositol 1,4,5-trisphosphate generation in intact cells and inositol 1,4,5-trisphosphate-induced Ca2+ release from microsomes, revealed the sustaining process of the waves to be discharge of Ca2+ from the inositol 1,4,5-trisphosphate- (and not the ryanodine-) sensitive stores distributed along the neurites. The activation of the cognate receptor appears to result from the coordinate action of the second messenger and Ca2+. Because of their properties and orientation, the waves could participate in the control of not only conventional cell activities, but also excitability and differential processing of inputs, and thus of electrochemical computation in nerve cells.

Computer-assisted registration, segmentation, and 3D reconstruction from images of neuronal tissue sections

Neuroscientists have studied the relationship between nerve cell morphology and function for over a century. To pursue these studies, they need accurate three-dimensional models of nerve cells that facilitate detailed anatomical measurement and the identification of internal structures. Although serial transmission electron microscopy has been a source of such models since the mid 1960s, model reconstruction and analysis remain very time consuming. The authors have developed a new approach to reconstructing and visualizing 3D nerve cell models from serial microscopy. An interactive system exploits recent computer graphics and computer vision techniques to significantly reduce the time required to build such models. The key ingredients of the system are a digital "blink comparator" for section registration, "snakes," or active deformable contours, for semiautomated cell segmentation, and voxel-based techniques for 3D reconstruction and visualization of complex cell volumes with internal structures.

The effect of in vitro and in vivo toluene exposure on rat erythrocyte and synaptosome membrane integral enzymes.

The mechanism of the anaesthetic effect of toluene on the central nervous system (CNS) was studied by using rat erythrocyte and synaptosome membranes as nerve cell models both in vitro and in vivo. The activities of the membrane-bound integral enzymes acetylcholinesterase (AChE), total adenosine triphosphatase (total ATPase) and magnesium-activated adenosine triphosphatase (Mg2+-ATPase) were determined. A short-term exposure to 2000 p.p.m. of toluene had an inhibitory effect on the enzyme activities studied. The degree of inhibition in erythrocyte membranes in vitro and in vivo, and in synaptosome membranes in vitro were in good correlation. In in vivo conditions, the synaptosome-bound enzymes were, however, significantly more inhibited by toluene, which indicates that membranes in vivo are even more vulnerable to the toxic effects of organic solvents than they are as isolated membranes in vitro. However, our results show that in vitro experiments can be used to predict the toxic nerve cell membrane effects of organic solvents. Toluene caused similar enzyme inhibitions both in neural cell membranes and in erythrocyte membranes. Thus, even peripheral non-excitable cell membranes, like erythrocytes, can be used as nerve cell membrane models in studies on the mechanism of the anaesthesia caused by solvents.

Changes in trichloroethylene-treated rat erythrocyte membranes in vitro.

Trichloroethylene (TCE) is a widely used organic solvent, the most important toxic effect of which is a narcotic central nervous (CNS) effect. In the present study we have used rat erythrocyte membranes as a nerve cell model for studying the changes in membrane integrity caused by TCE treatment. The parameters determined were osmotic resistance and the activities of acetylcholinesterase (AchE) and adenosinetriphosphatase (ATPase), both of which are integral membrane proteins. TCE had a dose-dependent effect on all these parameters. It increased the osmotic resistance at low concentrations and caused a decrease at high concentrations. Enzyme inhibition was only significant at high solvent concentrations. Decrease of temperature potentiated these effects. Our results indicate that changes in membrane proteins may be the initial factor leading to other changes in the membrane, e.g. increased osmotic resistance.

Modeling the electrical behavior of anatomically complex neurons using a network analysis program: passive membrane.

We describe the application of a popular and widely available electrical circuit simulation program called SPICE to modeling the electrical behavior of neurons with passive membrane properties and arbitrarily complex dendritic trees. Transient responses may be calculated at any location in the cell model following current, voltage or conductance perturbations at any point. A numbering method is described for binary trees which is helpful in transforming complex dendritic structures into a coded list of short cylindrical dendritic segments suitable for input to SPICE. Individual segments are modeled as isopotential compartments comprised of a parallel resistor and capacitor, representing the transmembrane impedance, in series with one or two core resistors. Synaptic current is modeled by a current source controlled by the local membrane potential and an "alpha-shaped" voltage, thus simulating a conductance change in series with a driving potential. Extensively branched test cell circuits were constructed which satisfied the equivalent cylinder constraints (Rall 1959). These model neurons were perturbed by independent current sources and by synaptic currents. Responses calculated by SPICE are compared with analytical results. With appropriately chosen model parameters, extremely accurate transient calculations may be obtained. Details of the SPICE circuit elements are presented, along with illustrative examples sufficient to allow implementation of passive nerve cell models on a number of common computers. Methods for modeling excitable membrane are presented in the companion paper (Bunow et al. 1985).

Firing behaviour in nerve cell models with a two-state pore system.

The firing behaviour of simple nerve cell models with a two-state pore system was investigated by computer simulation. The pores were assumed to open and close randomly, with the probabilities for opening an closing calculated from the equations found by Hodgkin and Huxley for the squid giant axon. The cell models had a cell body and an axon with an initial segment with a smaller diameter. Both the case of a uniform membrane with constant pore densities all over the cell, and the case of a cell body membrane with only a leakage conductance (a "passive" membrane) were investigated. The results indicate that the firing behaviour of a small nerve cell may be significantly influenced by the finite number of pores in the initial segment. In contrast to the original Hodgkin-Huxley equations which give a very non-linear frequency-current relationship in such nerve cell models, a fairly linear relationship over a large current range was found in many cases. It was estimated that the diameter of the initial segment must be less than approximately 1 micron and the length larger than half a space constant, in order to obtain a current frequency relationship significantly different from that predicted by the original Hodgkin-Huxley equations.

Experiments of Pattern Discriminating System Using Neural Cell Models

A simple pattern discriminating system using a nerve cell model as an analog threshold device is presented, in which a voltage controlled blocking oscillator (VCBO) is used as the electronic model of a nerve cell. The system is designed to discriminate the numeral patterns (0,1,...,9). A new analog decision-making neural network is used in the final stage to discriminate the pattern, and successful experimental results are shown.

Repetitive firing behavior in nerve cell models based upon a simplified form of the Hodgkin-Huxley equations

An investigation of a nerve cell model based upon a simplified form of the Hodgkin-Huxley equations has been carried through. Several different cases have been treated, and analytical solutions for the frequency-current relationship have been obtained for each case. The basic model is a nerve cell with a sharp threshold for firing, and where the steady state values of the membrane conductances are independent of the potential below threshold. The form of the frequency-current relationship was for this model given by f=[ln(1 + 1/ i)+ t 0] -1, where f is the firing frequency, i is the injected current, and t 0 is approximately the duration of the action potential. This relationship can be reasonably well fitted by a power law. The largest discrepancy is found around the current threshold for firing, where the above relationship has a vertical tangent. The effectupon this basic model of an additional dependence of the steady state values of the membrane conductances upon the membrane potential was investigated. It was found that an increase of the sodium conductance with the potential made the frequency-current relationship more linear, while an increase of the potassium conductance with the potential made the relationship less linear. This last case resembled more the frequency-current relationship for the squid giant axon investigated by Hodgkin and Huxley. The effects of a slow component of the potassium conductance and the effects of an electrogenic pump were also investigated. Both of these mechanisms resulted in adaptation. From the onset of a current step increase, the firing frequency rapidly increased with time and thereafter gradually decreased toward a steady state, non-zero value. Both of these adapting mechanisms made the frequency-current relationship more linear, and the forms of the steady state relationships were almost the same in the two cases. It will thus be difficult to decide between the two mechanisms possibly responsible for adaptation from the observed steady state frequency-current relationship alone.