Low dose isoflurane exerts opposing effects on neuronal network excitability in neocortex and hippocampus

The anesthetic excitement phase occurring during induction of anesthesia with volatile anesthetics is a well-known phenomenon in clinical practice. However, the physiological mechanisms underlying anesthetic-induced excitation are still unclear. Here we provide evidence from in vitro experiments performed on rat brain slices that the general anesthetic isoflurane at a concentration of about 0.1 mM can enhance neuronal network excitability in the hippocampus, while simultaneously reducing it in the neocortex. In contrast, isoflurane tissue concentrations above 0.3 mM expectedly caused a pronounced reduction in both brain regions. Neuronal network excitability was assessed by combining simultaneous multisite stimulation via a multielectrode array with recording intrinsic optical signals as a measure of neuronal population activity.     

Clinically Relevant Concentration Determination of Inhaled Anesthetics (Halothane,Isoflurane, Sevoflurane,and Desflurane) by <Superscript>19</Superscript>F NMR

Biophysical studies of protein–anesthetic interactions using nuclear magnetic resonance (NMR) spectroscopy are often conducted by the addition of micro amounts of neat inhaled anesthetic which yields much higher than clinically relevant (0.2–0.5 mM) anesthetic concentrations. We report a ¹⁹F NMR technique to measure clinically relevant inhaled anesthetic concentrations from saturated aqueous solutions of these anesthetics (halothane, isoflurane, sevoflurane, and desflurane). We use a setup with a 3-mm NMR tube (containing trifluoroacetic acid as standard), coaxially inserted in a 5-mm NMR tube containing anesthetic solution under investigation. All experiments are conducted in a 5-mm NMR probe. We also have provided standard curves for four inhaled anesthetics using NMR technique. The standard curve for each of these anesthetics is helpful in determining the prerequisite amount of aqueous anesthetic solution required to prepare clinically relevant concentrations for protein–anesthetic interaction studies. Parts of the results to be presented at Society for Neuroscience meeting, 2008.     

Effects of isoflurane concentration on basic echocardiographic parameters of the left ventricle in rats

Transthoracic echocardiography (TTE) has become an important modality for the assessment of cardiac structure and function in animal experiments. The acquisition of echocardiographic images in rats requires sedation/anesthesia to keep the rats immobile. Commonly used anesthetic regimens include intraperitoneal or inhalational application of various anesthetics. Several studies have compared the effects of anesthetic agents on echocardiographic parameters in rats; however, none of them examined the effects of different concentrations of inhalational anesthetics on echocardiographic parameters. Accordingly, the aim of this study was to examine the effects of different concentrations of isoflurane used for anesthesia during TTE examination in rats on basic echocardiographic parameters of left ventricular (LV) anatomy and systolic function. TTE examinations were performed in adult male Wistar rats (n=10) anesthetized with isoflurane at concentrations of 1.5-3 %. Standard echocardiograms were recorded for off-line analysis. An absence of changes in basic echocardiographic parameters of LV anatomy and systolic function was found under isoflurane anesthesia using concentrations between 1.5-2.5 %. An isoflurane concentration of 3 % caused a small, but statistically significant, increase in LV chamber dimensions without a concomitant change in heart rate or fractional shortening. For the purpose of TTE examination in the rat, our results suggest that isoflurane concentrations 相似文献   

Correlation of Electrical and Permeability Properties of Ion-Selective Membranes

The linear phenomenological equations giving particle and practical fluxes of a single electrolyte across an ion-selective membrane are stated and interrelated. It is shown that the experimental measurements commonly made in biological and synthetic membrane studies may be used, with minor modification, to obtain the phenomenological transport coefficients and their concentration dependences. It is demonstrated that the electrical properties of a homogeneous membrane may be obtained as functions of the bathing solution concentration by combining fluxes measured under open and short circuit. Attention is paid to the use of radiotracers when measuring ionic fluxes. To obtain all the phenomenological coefficients at least one measurement must be made under a pressure gradient. The experimental difficulties in such measurements are discussed and the merits and demerits of various experiments considered. The problems of measuring potentials and concentrations at the low pressure face of a supported membrane make several mathematically simple approaches experimentally unattractive. The best methods appear to be either the measurement of a succession of “apparent osmotic pressures” under concentration differences sufficiently small that the membrane does not require support or the study of “reverse osmosis”. Sets of equations are given which enable the phenomenological coefficients to be evaluated from convenient experiments. With a stable homogeneous membrane nine coefficients may be obtained thus enabling either the applicability of the reciprocal relations or the applicability of linear theory under the conditions of the experiments to be tested. For a discontinuous system the six independent coefficients may be obtained from experiments in a single membrane cell.     

Binding of volatile anesthetics to serum albumin: measurements of enthalpy and solvent contributions

This study directly examines the enthalpic contributions to binding in aqueous solution of closely related anesthetic haloethers (desflurane, isoflurane, enflurane, and sevoflurane), a haloalkane (halothane), and an intravenous anesthetic (propofol) to bovine and human serum albumin (BSA and HSA) using isothermal titration calorimetry. Binding to serum albumin is exothermic, yielding enthalpies (DeltaH(obs)) of -3 to -6 kcal/mol for BSA with a rank order of apparent equilibrium association constants (K(a) values): desflurane > isoflurane approximately enflurane > halothane >or= sevoflurane, with the differences being largely ascribed to entropic contributions. Competition experiments indicate that volatile anesthetics, at low concentrations, share the same sites in albumin previously identified in crystallographic and photo-cross-linking studies. The magnitude of the observed DeltaH increased linearly with increased reaction temperature, reflecting negative changes in heat capacities (DeltaC(p)). These -DeltaC(p) values significantly exceed those calculated for burial of each anesthetic in a hydrophobic pocket. The enhanced stabilities of the albumin/anesthetic complexes and -DeltaC(p) are consistent with favorable solvent rearrangements that promote binding. This idea is supported by substitution of D(2)O for H(2)O that significantly reduces the favorable binding enthalpy observed for desflurane and isoflurane, with an opposing increase of DeltaS(obs). From these results, we infer that solvent restructuring, resulting from release of water weakly bound to anesthetic and anesthetic-binding sites, is a dominant and favorable contributor to the enthalpy and entropy of binding to proteins.     

Glucose diffusion in pancreatic islets of Langerhans.

We investigate the time required for glucose to diffuse through an isolated pancreatic islet of Langerhans and reach an equilibrium. This question is relevant in the context of in vitro electrophysiological studies of the response of an islet to step changes in the bath glucose concentration. Islet cells are electrically coupled by gap junctions, so nonuniformities in islet glucose concentration may be reflected in the activity of cells on the islet periphery, where electrical recordings are made. Using a mathematical model of hindered glucose diffusion, we investigate the effects of the islet porosity and the permeability of a surrounding layer of acinar cells. A major factor in the determination of the equilibrium time is the transport of glucose into islet beta-cells, which removes glucose from the interstitial spaces where diffusion occurs. This transport is incorporated by using a model of the GLUT-2 glucose transporter. We find that several minutes are required for the islet to equilibrate to a 10 mM change in bath glucose, a typical protocol in islet experiments. It is therefore likely that in electrophysiological islet experiments the glucose distribution is nonuniform for several minutes after a step change in bath glucose. The delay in glucose penetration to the inner portions of the islet may be a major contributing factor to the 1-2-min delay in islet electrical activity typically observed after bath application of a stimulatory concentration of glucose.     

Differential effects of propofol and isoflurane on glucose utilization and insulin secretion

AimsVolatile anesthetics, such as isoflurane, reverse glucose-induced inhibition of pancreatic adenosine triphosphate-sensitive potassium (K_ATP) channel activity, resulting in reduced insulin secretion and impaired glucose tolerance. No previous studies have investigated the effects of intravenous anesthetics, such as propofol, on pancreatic K_ATP channels. We investigated the cellular mechanisms underlying the effects of isoflurane and propofol on pancreatic K_ATP channels and insulin secretion.Main methodsIntravenous glucose tolerance tests (IVGTT) were performed on male rabbits. Pancreatic islets were isolated from male rats and used for a perifusion study, measurement of intracellular ATP concentration ([ATP]_i), and patch clamp experiments.Key findingsGlucose stimulus significantly increased insulin secretion during propofol anesthesia, but not isoflurane anesthesia, in IVGTT study. In perifusion experiments, both islets exposed to propofol and control islets not exposed to anesthetic had a biphasic insulin secretory response to a high dose of glucose. However, isoflurane markedly inhibited glucose-induced insulin secretion. In a patch clamp study, the relationship between ATP concentration and channel activity could be fitted by the Hill equation with a half-maximal inhibition of 22.4, 15.8, and 218.8 μM in the absence of anesthetic, and with propofol, and isoflurane, respectively. [ATP]_i and single K_ATP channel conductance did not differ in islets exposed to isoflurane or propofol.SignificanceOur results indicate that isoflurane, but not propofol, decreases the ATP sensitivity of K_ATP channels and impairs glucose-stimulated insulin release. These differential actions of isoflurane and propofol on ATP sensitivity may explain the differential effects of isoflurane and propofol on insulin release.     

Comparison of the effects of volatile anesthetics in varying concentrations on brain energy metabolism with brain ischemia in rats

The effects of varying concentrations and types of volatile anesthetics on neurochemical sequelae of brain ischemia were evaluated in the rat. Rats were assigned to treatment defined by a 3×3 design (anesthetic type and dose) with 5 rats/cell. Each group received halothane, enflurane, or isoflurane 0.5, 1.0, or 2.0 MAC (minimal alevolar concentration). This was followed by preischemic plasma glucose sampling, 5 min hypotension (30 mmHg) and 5 min decapitation cerebral ischemia. Preischemia plasma glucose increased with increasing anesthetic concentration and was highest in the isoflurane groups, varying from a low (±SD) of 7.19±1.79 mol/ml in the 0.5 MAC halothane group to a high of 12.68±3.65 mol/ml in the 2.0 MAC isoflurane group. End-ischemic brain lactate correlated with preischemic plasma glucose (r=0.5, =0.5). We conclude that increasing concentration of volatile anesthesia with iv phenylephrine blood pressure support produces higher levels of plasma glucose and brain lactate with cerebral ischemia.     

The action of local anesthetics on myelin structure and nerve conduction in toad sciatic nerve

X-ray scattering and electrophysiological experiments were performed on toad sciatic nerves in the presence of local anesthetics. In vitro experiments were performed on dissected nerves superfused with Ringer's solutions containing procaine, lidocaine, tetracaine, or dibucaine. In vivo experiments were performed on nerves dissected from animals anesthesized by targeted injections of tetracaine-containing solutions. In all cases the anesthetics were found to have the same effects on the x-ray scattering spectra: the intensity ratio of the even-order to the odd-order reflections increases and the lattice parameter increases. These changes are reversible upon removal of the anesthetic. The magnitude of the structural changes varies with the duration of the superfusion and with the nature and concentration of the anesthetic molecule. A striking quantitative correlation was observed between the structural effects and the potency of the anesthetic. Electron density profiles, which hardly showed any structural alteration of the unit membrane, clearly indicated that the anesthetics have the effect of moving the pairs of membranes apart by increasing the thickness of the cytoplasmic space. Electrophysiological measurements performed on the very samples used in the x-ray scattering experiments showed that the amplitude of the compound action potential is affected earlier than the structure of myelin (as revealed by the x-ray scattering experiments), whereas conduction velocity closely follows the structural alterations.     

Bayesian analysis of serial dilution assays

In a serial dilution assay, the concentration of a compound is estimated by combining measurements of several different dilutions of an unknown sample. The relation between concentration and measurement is nonlinear and heteroscedastic, and so it is not appropriate to weight these measurements equally. In the standard existing approach for analysis of these data, a large proportion of the measurements are discarded as being above or below detection limits. We present a Bayesian method for jointly estimating the calibration curve and the unknown concentrations using all the data. Compared to the existing method, our estimates have much lower standard errors and give estimates even when all the measurements are outside the "detection limits." We evaluate our method empirically using laboratory data on cockroach allergens measured in house dust samples. Our estimates are much more accurate than those obtained using the usual approach. In addition, we develop a method for determining the "effective weight" attached to each measurement, based on a local linearization of the estimated model. The effective weight can give insight into the information conveyed by each data point and suggests potential improvements in design of serial dilution experiments.     

19F nuclear magnetic resonance investigation of stereoselective binding of isoflurane to bovine serum albumin.

Whether proteins or lipids are the primary target sites for general anesthetic action has engendered considerable debate. Recent in vivo studies have shown that the S(+) and R(-) enantiomers of isoflurane are not equipotent, implying involvement of proteins. Bovine serum albumin (BSA), a soluble protein devoid of lipid, contains specific binding sites for isoflurane and other anesthetics. We therefore conducted 19F nuclear magnetic resonance measurements to determine whether binding of isoflurane to BSA was stereoselective. Isoflurane chemical shifts were measured as a function of BSA concentration to determine the chemical shift differences between the free and bound isoflurane. KD was determined by measuring the 19F transverse relaxation times (T2) as a function of isoflurane concentration. The binding duration was determined by assessing increases in 1/T2 as a result of isoflurane exchanging between the free and bound states. The S(+) and R(-) enantiomers exhibited no stereoselectivity in chemical shifts and KD values (KD = 1.3 +/- 0.2 mM, mean +/- SE, for S(+), R(-), and the racemic mixture). Nonetheless, stereoselectivity was observed in dynamic binding parameters; the S(+) enantiomer bound with slower association and dissociation rates than the R(-).     

Isoflurane Reversibly Destabilizes Hippocampal Dendritic Spines by an Actin-Dependent Mechanism

General anesthetics produce a reversible coma-like state through modulation of excitatory and inhibitory synaptic transmission. Recent evidence suggests that anesthetic exposure can also lead to sustained cognitive dysfunction. However, the subcellular effects of anesthetics on the structure of established synapses are not known. We investigated effects of the widely used volatile anesthetic isoflurane on the structural stability of hippocampal dendritic spines, a postsynaptic structure critical to excitatory synaptic transmission in learning and memory. Exposure to clinical concentrations of isoflurane induced rapid and non-uniform shrinkage and loss of dendritic spines in mature cultured rat hippocampal neurons. Spine shrinkage was associated with a reduction in spine F-actin concentration. Spine loss was prevented by either jasplakinolide or cytochalasin D, drugs that prevent F-actin disassembly. Isoflurane-induced spine shrinkage and loss were reversible upon isoflurane elimination. Thus, isoflurane destabilizes spine F-actin, resulting in changes to dendritic spine morphology and number. These findings support an actin-based mechanism for isoflurane-induced alterations of synaptic structure in the hippocampus. These reversible alterations in dendritic spine structure have important implications for acute anesthetic effects on excitatory synaptic transmission and synaptic stability in the hippocampus, a locus for anesthetic-induced amnesia, and have important implications for anesthetic effects on synaptic plasticity.     

Preliminary comments on a redescription of Pseudolithophyllum fuegianum and Titanoderma conspectum (Rhodophyta,Corallinales) in Tierra del Fuego,Argentina

The effect of twelve drugs and chemical compounds on the narcosis of Brachionus plicatilis was studied using standardized laboratory conditions. Drug efficacy was compared by calculating EC₅₀ (effective concentration causing narcosis in 50% of animals), time necessary to reach narcosis in 50% of animals, concentration range of activity, and degree of extension after preservation. The local anesthetic Bupivacaine was found to be most effective by all criteria. Our previous data and preliminary field experiments indicated that drug sensitivity varies widely, even between congeneric taxa. The anesthetic effect of carbonated water was also investigated.     

Molecular understanding of Abeta peptide interaction with isoflurane, propofol, and thiopental: NMR spectroscopic study

Abeta peptide is the major component of senile plaques (SP), which accumulate in the brain of a patient with Alzheimer's disease (AD). A recent report indicated that isoflurane enhanced Abeta oligomerization (micro-aggregation) and subsequent cytotoxicity of the Abeta peptide. A separate study showed that a clinically relevant concentration of isoflurane induces apoptosis and increases Abeta production in a human neuroglioma cell line. In vitro studies have indicated that halothane interacts specifically with Abeta peptide to induce oligomerization and that Abeta42 oligomerizes faster than Abeta40. The specific interactions of isoflurane, propofol, and thiopental with uniformly 15N labeled Abeta40 and Abeta42 peptide were investigated using multidimensional nuclear magnetic resonance (NMR) experiments. We found that isoflurane and propofol (at higher concentration) interact with Abeta40 peptides and induce Abeta oligomerization. Thiopental does not interact with specific residues (G29, A30, and I31) of Abeta40; hence, the peptide remains in the monomeric form. On the basis of our NMR study, thiopental does not oligomerize Abeta40 even at higher concentrations.     

Relationship of feline bispectral index to multiples of isoflurane minimum alveolar concentration

The study reported here was done to determine the relationship between bispectral index (BIS) values and minimum alveolar concentration (MAC) multiples of isoflurane in cats. Isoflurane MAC was determined using the tail-clamp method in eight domestic cats. Ten days later, the cats were anesthetized a second time with isoflurane at each of five MAC multiples administered in random order. Ventilation was controlled and, after a 20-min equilibration period at each MAC multiple of isoflurane, BIS data were collected for 5 min and the median BIS value calculated. Data from each isoflurane MAC multiple were compared using analysis of variance for repeated measures, and statistical significance was set at P < 0.05. The MAC of isoflurane (mean +/- 1 standard deviation) was 1.8% +/- 0.2%. BIS values at 0.5 MAC could not be recorded due to spontaneous movement in all eight cats. BIS values at 2.0 MAC were confounded by burst suppression in seven of the eight cats. Over the range of 0.8 to 1.5 MAC, BIS values decreased significantly with increasing end-tidal isoflurane concentrations. Mean (+/- 1 standard deviation) BIS measurements were 32 +/- 3 at 0.8 MAC, 20 +/- 4 at 1.0 MAC, and 5 +/- 3 at 1.5 MAC. Therefore, BIS values are inversely and linearly related to end-tidal isoflurane concentrations in anesthetized cats. However, the consistently low BIS values recorded in this study suggest that clinical BIS endpoints used to titrate anesthetic agents in humans may not be applicable to cats.     

Estimation of minimum alveolar concentration (MAC) for halothane, enflurane and isoflurane in spontaneously breathing guinea pigs

MAC for halothane, enflurane and isoflurane was determined in guinea pigs (Cavia porcellus) exposed to constant anesthetic concentrations (2.5 hours each) in a flow-through glass chamber. The following values were obtained (N = 8 for each anesthetic): 1.01 +/- 0.03 vol% for halothane, 2.17 +/- 0.04 vol% for enflurane, and 1.15 +/- 0.05 vol% for isoflurane. In guinea pigs, MAC for halothane and enflurane are similar to those reported for other rodents, while MAC for isoflurane is lower. The data indicate that guinea pigs possibly are more susceptible to isoflurane's anesthetic actions than other rodents.     

In Vitro Induction of Endothelial Apoptosis of the Post-Hypoxic Blood-Brain Barrier by Isoflurane but Not by Sevoflurane and Midazolam

Background

The effects of anesthetics on the injured brain continue to be the subject of controversial discussion. Since isoflurane has recently been shown to induce apoptosis of cerebral endothelial cells, this study compared different anesthetic compounds regarding their potential to induce cerebro-vascular apoptosis.

Methods

The in vitro model of the blood-brain barrier used in this study consisted of astrocyte-conditioned human umbilical vein endothelial cells (AC-HUVEC) has been used. After 24 h of deep hypoxia and reoxygenation or control treatment, AC-HUVEC were exposed to 0, 0.5, 1.0, or 2.0 times the minimum alveolar concentration of isoflurane or sevoflurane, or 0, 75, 150, or 300 nM of midazolam for 2 h. After 24 h, AC-HUVEC were harvested, and the degree of apoptosis was assessed by means of Western blots for the Bax and Bcl-2 ratio and, for controls and the highest concentration groups, terminal deoxynucleotidyl-mediated dUTP-biotin nick end labeling (TUNEL).

Results

Without hypoxic pretreatment, 2.0 MAC of isoflurane slightly increased TUNEL intensity compared to control and sevoflurane, but without any significant changes in the Bax and Bcl-2 ratio. After hypoxic pretreatment, exposure to isoflurane led to a multifold increase in the Bax and Bcl-2 ratio in a dose dependent manner, which was also significantly higher than the ratio observed in the 2 MAC sevoflurane group. TUNEL intensity in the post-hypoxic 2 MAC isoflurane group was increased by a factor of 11 vs. control and by 40 vs. sevoflurane. Sevoflurane and midazolam did not significantly alter these markers of apoptosis, when compared to the control group.

Conclusions

Isoflurane administered after hypoxia elevates markers of apoptosis in endothelial cells transdifferentiated to the cerebro-vascular endothelium. Endothelial apoptosis may be a previously underestimated mechanism of anesthetic neurotoxicity. Administration of high concentrations of isoflurane in experimental settings may have negative effects on the blood-brain barrier.     

A study of the "outward potassium channel" (OPC) in the frog oocyte. II. A study of the "inside-out" configuration

The single K-channel current reported in a previous note was also studied in "outside-out" conditions. The electrode filling solutions used for the "cell-attached" experiments faced in this case the intracellular side of the membrane patches, the extracellular side facing the bath saline, i.e. Ringer standard. The most significant observations were obtained with filling solutions with varying proportions in K/Na concentrations solutions. In the absence of Na+ ([K+] = 110 mM), the elementary conductance was still around 90 pS and the I/V diagram was again somewhat bell shaped, though the distinctive reduction of the elementary conductance began at more positive potentials (+110 mV). No inward current could be detected upon membrane repolarization also in this case. The rectification became less evident and conductance increased with increasing Na+ concentration in the filling solution, until the I/V curve became a linear one and conductance was 270 pS with standard Ringer. Distinct inward elementary currents were evident upon repolarization in these conditions. Thus a complex interaction between Na+ and K+ takes place for conduction through the outward K channel in the frog oocyte, both cations probably competing for at least one active site inside. Another interesting observation concerns the process of gating of the OPC: the open times of the elementary currents were in fact much greater in outside out experiments as compared to cell-attached experiments, probably due to the presence of Ca++ in contact with the inner membrane side. Even increasing Na+ concentration prolonged the open time duration. The gating of the OPC in the membrane was not only voltage dependent, but also Ca++ and Na+ dependent.     

The effect of isoflurane on erythrocyte membranes studied by ATR-FTIR

The effect of isoflurane on erythrocyte membranes has been investigated by means of attenuated total reflection infrared spectroscopy. Infrared spectra were measured on sonicated erythrocyte ghosts layered upon a ZnSe crystal covered with D(2)O saline solutions containing increasing amounts of isoflurane. At clinically relevant anesthetic concentrations and 37 degrees C, significant changes in the structural and dynamic properties of the membrane phospholipid bilayers are observed. Both the acyl chain methylene symmetric and asymmetric stretching modes and the carbonyl ester stretching band displayed frequency shifts interpreted as transitions toward disordered liquid-like structure accompanied by dehydration of the phospholipid polar heads. In turn, no secondary structure-linked changes are observed in the amide I region of membrane proteins. Higher anesthetic concentrations (500-900 microM), resulted in progressive detachment of the multilayers from the ATR crystal and irreversible formation of denatured protein. Polarization studies in correspondence of the acyl lipid methylene stretching bands indicated that isoflurane decreases the dichroic ratio thus inducing disorder in the orientation of the lipid acyl chains.     

Kinetics of cellulose regeneration from cellulose--NaOH--water gels and comparison with cellulose--N-methylmorpholine-N-oxide--water solutions

The regeneration kinetics of cellulose from cellulose--NaOH--water gels immersed in a nonsolvent bath is studied in detail. Cellulose concentration, bath type, and temperature were varied, and diffusion coefficients were determined. The results were compared with data measured and taken from the literature on the regeneration kinetics of cellulose from cellulose--N-methylmorpholine-N-oxide (NMMO) monohydrate solutions. Different theories developed for the transport behavior of solutes in hydrogels or in porous media were tested on the systems studied. While the diffusion of NaOH from cellulose--NaOH--water gels into water has to be described with "porous media" approaches, the interpretation of NMMO diffusion is complicated because of the change of NMMO's state during regeneration (from solid crystalline to liquid) and the high concentration of NMMO in the sample. The activation energies were calculated from diffusion coefficient dependence on temperature for both systems and compared with the ones obtained from the rheological measurements. The activation energy of cellulose--NaOH--water systems does not depend on cellulose concentration or the way of measurement. This result shows that whatever the system is, pure NaOH--water solution, cellulose--NaOH--water solution, or cellulose--NaOH--water gel, it is NaOH hydrate with or without cellulose in solution, which is moving in the system. The swelling of cellulose in different nonsolvent liquids such as water or different alcohols during regeneration was investigated and interpreted using the Hildebrand parameter.