Isoflurane attenuates sepsis-associated lung injury.

Acute lung injury is one of major complications associated with sepsis, responsible for morbidity and mortality. Patients who suffer from acute lung injury often require respiratory support under sedations, and it would be important to know the role of sedatives in lung injury. We examined volatile anesthetic isoflurane, which is commonly used in surgical setting, but also used as an alternative sedative in intensive care settings in European countries and Canada. We found that isoflurane exposure attenuated neutrophil recruitment to the lungs in mice suffering from experimental polymicrobial abdominal sepsis. We found that isoflurane attenuated one of major neutrophil chemoattractants LTB4 mediated response via its receptor BLT1 in neutrophils. Furthermore, we have shown that isoflurane directly bound to BLT1 by a competition assay using newly developed labeled BLT1 antagonist, suggesting that isoflurane would be a BLT1 antagonist.

Volatile anesthetic gas concentration sensing using flow sensor fusion for use in Austere settings.

Flow sensors are often sensitive to the presence of volatile anesthetics. However, this sensitivity provides a unique opportunity to combine flow sensors of differing technological principles as an alternative to measuring volatile anesthetic gas concentration, particularly for austere settings. To determine the feasibility of flow sensor fusion for volatile anesthetic concentrations monitoring, eight flow sensors were tested with isoflurane, sevoflurane, and desflurane, ranging in concentrations from 0-4.5%, 0-3.5%, and 0-18%, respectively. Pairs of flow sensors were fit to the volatile anesthetic gas concentration with a leave-one-out cross-validation method to reduce the likelihood of overfitting. Bland-Altman was used for the final evaluation of sensor pair performance. Several sensor pairs yielded limits of agreement comparable to the rated accuracy of a commercial infrared spectrometer. The ultrasonic and orifice-plate flowmeters yielded the most combinations of viable sensor pairs for all three volatile anesthetic gases. Conclusion: Measuring volatile anesthetic gases using flow sensor fusion is a feasible low-cost, low-maintenance alternative to infrared spectroscopy. In this study, testing was done under steady-state conditions in 100% oxygen. Further testing is necessary to ensure sensor fusion performance under conditions that are more reflective of the clinical use case.

Anesthetics isoflurane and sevoflurane attenuate flagellin-mediated inflammation in the lung.

Isoflurane and sevoflurane are volatile anesthetics (VA) widely used in clinical practice to provide general anesthesia. We and others have previously shown that VAs have immunomodulatory effects and may have a significant impact on the progression of disease states. Flagellin is a component of Gram negative bacteria and plays a significant role in the pathophysiology of bacterial pneumonia through its binding to Toll-like Receptor 5 (TLR5). Our results showed that VAs, not an intravenous anesthetic, significantly attenuated the activation of TLR5 and the release of the neutrophil chemoattractant IL-8 from lung epithelial cells. Furthermore, flagellin-induced lung injury was significantly attenuated by VAs by inhibiting neutrophil migration to the bronchoalveolar space. The lungs of cystic fibrosis (CF) patients are highly colonized by Pseudomonas aeruginosa, which causes inflammation. The retrospective study of oxygenation in patients with CF who had received VA versus intravenous anesthesia suggested that VAs might have the protective effect for gas exchange. To understand the interaction between VAs and TLR5, a docking simulation was performed, which indicated that isoflurane and sevoflurane docked into the binding interphase between TLR5 and flagellin.

The effect of anesthetics on toll like receptor 9.

Toll like receptors (TLRs) are critical receptors to respond to danger signals, and their functions are relevant in the perioperative period. We previously reported that volatile anesthetics directly bound to TLR2 and TLR4 and attenuated their functions. Given that TLR9 can respond to mitochondrial DNA, a danger signal that is released upon tissue injury, we examined the role of anesthetics on TLR9 function. Our reporter assay showed that volatile anesthetics isoflurane and sevoflurane increased the activation of TLR9, while propofol attenuated it. TLR9 activation occurs via its dimerization. The dimerization is facilitated by unmethylated cytosine-phosphate-guanine (CpG) DNA as well as DNA containing cytosine at the second position from 5'-end (5'-xCx DNA). Our structural analysis using photoactivable anesthetics and rigid docking simulation showed that isoflurane and sevoflurane bound to both TLR9 dimer interface and 5'-xCx DNA binding site. Propofol bound to the TLR9 antagonist binding site. This is the first illustration that anesthetics can affect the binding of nucleic acids to their receptor. This study sets the foundation for the effect of anesthetics on TLR9 and will pave the way for future studies to determine the significance of such interactions in the clinical setting.

Isoflurane increases cell membrane fluidity significantly at clinical concentrations.

There is an on-going debate whether anesthetic drugs, such as isoflurane, can cause meaningful structural changes in cell membranes at clinical concentrations. In this study, the effects of isoflurane on lipid membrane fluidity were investigated using fluorescence anisotropy and spectroscopy. In order to get a complete picture, four very different membrane systems (erythrocyte ghosts, a 5-lipid mixture that mimics brain endothelial cell membrane, POPC/Chol, and pure DPPC) were selected for the study. In all four systems, we found that fluorescence anisotropies of DPH-PC, nile-red, and TMA-DPH decrease significantly at the isoflurane concentrations of 1 mM and 5 mM. Furthermore, the excimer/monomer (E/M) ratio of dipyrene-PC jumps immediately after the addition of isoflurane. We found that isoflurane is quite effective to loosen up highly ordered lipid domains with saturated lipids. Interestingly, 1 mM isoflurane causes a larger decrease of nile-red fluorescence anisotropy in erythrocyte ghosts than 52.2 mM of ethanol, which is three times the legal limit of blood alcohol level. Our results paint a consistent picture that isoflurane at clinical concentrations causes significant and immediate increase of membrane fluidity in a wide range of membrane systems.

[Climate footprint of halogenated inhalation anesthetics]. / Klimateffekterna från anestesin kan minska.

This study estimated the climate footprint of halogenated inhalation anesthetics in Sweden and estimated effects of a decreased use of these compounds. We collected data on sales of desflurane, sevoflurane and isoflurane in Sweden during 2017 and calculated the mass of CO2 equivalents (CO2e) using Global Warming Potential data over 100 years for the compounds. Inhalation anesthetics contributed by 5000 tons of CO2e which corresponds to 0.005 percent of the Swedish climate footprint. By replacing desflurane with sevoflurane the footprint can be reduced by 73 percent. By replacing sevoflurane with intravenous propofol the climate effect can be reduced further by at least 2 orders of magnitude.

Recent advances in the neuroprotective effects of medical gases.

Central nervous system injuries are a leading cause of death and disability worldwide. Although the exact pathophysiological mechanisms of various brain injuries vary, central nervous system injuries often result in an inflammatory response, and subsequently lead to brain damage. This suggests that neuroprotection may be necessany in the treatment of multiple disease models. The use of medical gases as neuroprotective agents has gained great attention in the medical field. Medical gases include common gases, such as oxygen, hydrogen and carbon dioxide; hydrogen sulphide and nitric oxide that have been considered toxic; volatile anesthetic gases, such as isoflurane and sevoflurane; and inert gases like helium, argon, and xenon. The neuroprotection from these medical gases has been investigated in experimental animal models of various types of brain injuries, such as traumatic brain injury, stroke, subarachnoid hemorrhage, cerebral ischemic/reperfusion injury, and neurodegenerative diseases. Nevertheless, the transition into the clinical practice is still lagging. This delay could be attributed to the contradictory paradigms and the conflicting results that have been obtained from experimental models, as well as the presence of inconsistent reports regarding their safety. In this review, we summarize the potential mechanisms underlying the neuroprotective effects of medical gases and discuss possible candidates that could improve the outcomes of brain injury.

Kinetics of isoflurane and sevoflurane in a unidirectional displacement flow and the relevance to anesthetic gas exposure by operating room personnel.

International guidelines recommend the use of ventilation systems in operating rooms to reduce the concentration of potentially hazardous substances such as anesthetic gases. The exhaust air grilles of these systems are typically located in the lower corners of the operating room and pick up two-thirds of the air volume, whereas the final third is taken from near the ceiling, which guarantees an optimal perfusion of the operating room with a sterile filtered air supply. However, this setup is also employed because anesthetic gases have a higher molecular weight than the components of air and should pool on the floor if movement is kept to a minimum and if a ventilation system with a unidirectional displacement flow is employed. However, this anticipated pooling of volatile anesthetics at the floor level has never been proven. Thus, we herein investigated the flow behaviors of isoflurane, sevoflurane, and carbon dioxide (for comparison) in a measuring chamber sized 2.46 × 1.85 × 5.40 m with a velocity of 0.3 m/sec and a degree of turbulence <20%. Gas concentrations were measured at 1,728 measuring positions throughout the measuring chamber, and the flow behaviors of isoflurane and sevoflurane were found to be similar, with an overlap of 90%. The largest spread of both gases was 55 cm at 5.4 m from the emission source. Interestingly, neither isoflurane nor sevoflurane was detected at floor level, but a continuous cone-like spreading was observed due to gravity. In contrast, carbon dioxide accumulated at floor level in the form of a gas cloud. Thus, floor level exhaust ventilation systems are likely unsuitable for the collection and removal of anesthetic gases from operating rooms.

Enantioselective capillary electrophoresis for pharmacokinetic analysis of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in equines anesthetized with ketamine and isoflurane.

An enantioselective assay for the determination of methadone and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in equine plasma based on capillary electrophoresis with highly sulfated γ-cyclodextrin as chiral selector and electrokinetic analyte injection is described. The assay is based on liquid/liquid extraction of the analytes at alkaline pH from 0.1 mL plasma followed by electrokinetic sample injection of the analytes from the extract across a buffer plug without chiral selector. Separation occurs cationically at normal polarity in a pH 3 phosphate buffer containing 0.16% (w/v) of highly sulfated γ-cyclodextrin. The developed assay is precise (intra- and interday RSD < 4% and < 7%, respectively), is capable to determine enantiomer levels of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in plasma down to 2.5 ng/mL, and was successfully applied to monitor enantiomer drug and metabolite levels in plasma of a pony that was anesthetized with racemic ketamine and isoflurane and received a bolus of racemic methadone and a bolus followed by constant rate infusion of racemic methadone. The data suggest that the assay is well suited for pharmacokinetic purposes.

Investigation of Anesthetic-Protein Interactions by a Thermodynamic Approach.

Anesthetics can interact with a wide variety of proteins in the body, including ion channels and alter their activity, but little is known about the molecular mechanisms of the interactions responsible for the functional activity. Characterization of the nature of anesthetic-protein interactions therefore is important and requires the complete analysis of the binding energetics. Isothermal titration calorimetry (ITC) is the only technique that allows quantitative determination of all thermodynamic parameters, including the equilibrium binding constant (KB), the standard Gibbs free energy change (ΔG), the enthalpy change (ΔH), the entropy change (ΔS), heat capacity change (ΔCp), and stoichiometry (n) of the reaction. ITC does not require any labeling or modification of the interacting partners analyzed and can be performed in solution with small amounts of reagents. In this chapter we describe the general properties of the ITC method, highlighting some critical aspects of experimental planning and data analysis, with practical application to anesthetic-protein interactions.

X-Ray Crystallographic Studies for Revealing Binding Sites of General Anesthetics in Pentameric Ligand-Gated Ion Channels.

X-ray crystallography is a powerful tool in structural biology and can offer insight into structured-based understanding of general anesthetic action on various relevant molecular targets, including pentameric ligand-gated ion channels (pLGICs). In this chapter, we outline the procedures for expression and purification of pLGICs. Optimization of crystallization conditions, especially to achieve high-resolution structures of pLGICs bound with general anesthetics, is also presented. Case studies of pLGICs bound with the volatile general anesthetic isoflurane, 2-bromoethanol, and the intravenous general anesthetic ketamine are revisited.

Structural Analysis of Anesthetics in Complex with Soluble Proteins.

Anesthetics interact with a broad range of different targets, including both soluble and membrane-bound proteins. Understanding these interactions at the molecular level requires detailed structural knowledge of anesthetic-protein complexes, and one of the most productive routes to such knowledge is X-ray crystallography. In this chapter we discuss the application of this technique to the analysis of complexes of anesthetics with soluble proteins. The model protein apoferritin is highlighted, and protocols are presented for obtaining diffraction-quality crystals of this protein in complex with different general anesthetics.

Solution NMR Studies of Anesthetic Interactions with Ion Channels.

NMR spectroscopy is one of the major tools to provide atomic resolution protein structural information. It has been used to elucidate the molecular details of interactions between anesthetics and ion channels, to identify anesthetic binding sites, and to characterize channel dynamics and changes introduced by anesthetics. In this chapter, we present solution NMR methods essential for investigating interactions between ion channels and general anesthetics, including both volatile and intravenous anesthetics. Case studies are provided with a focus on pentameric ligand-gated ion channels and the voltage-gated sodium channel NaChBac.

Molecular basis for potentiation of Cx36 gap junction channel conductance by n-alcohols and general anesthetics.

In our recent study, we have demonstrated that short carbon chain n-alcohols (up to octanol) stimulated while long carbon chain n-alcohols inhibited the conductance of connexin (Cx) 36 (Cx36) gap junction (GJ) channels. In contrast, GJ channels composed of other types of Cxs all were inhibited by n-alcohols independent of their carbon chain length. To identify the putative structural domains of Cx36, responsible for the dual effect of n-alcohols, we performed structural modeling of Cx36 protein docking with hexanol and isoflurane that stimulated as well as nonanol and carbenoxolone that inhibited the conductance of Cx36 GJs and revealed their multiple common docking sites and a single pocket accessible only to hexanol and isoflurane. The pocket is located in the vicinity of three unique cysteine residues, namely C264 in the fourth, and C92 and C87 in the second transmembrane domain of the neighboring Cx36 subunits. To examine the hypothesis that disulphide bonding might be involved in the stimulatory effect of hexanol and isoflurane, we generated cysteine substitutions in Cx36 and demonstrated by a dual whole-cell patch-clamp technique that in HeLa (human cervix carcinoma cell line) and N2A (mouse neuroblastoma cell line) cells these mutations reversed the stimulatory effect of hexanol and isoflurane to inhibitory one, typical of other Cxs that lack respective cysteines and a specific docking pocket for these compounds. Our findings suggest that the stimulatory effect of hexanol and isoflurane on Cx36 GJ conductance could be achieved by re-shuffling of the inter-subunit disulphide bond between C264 and C92 to the intra-subunit one between C264 and C87.

Path to the Desensitized State of Ligand-Gated Ion Channels: Why Are Inhibitory and Excitatory Receptors Different?

The family of pentameric ligand-gated ion channels (pLGICs) includes both inhibitory and excitatory receptors. Electrophysiological methods have explored the time-dependent ion currents induced by their neurotransmitter agonists. Kinetic modeling requires a minimum of three conformational states: resting, active, and desensitized. However, current traces of inhibitory and excitatory pLGICs differ substantially. Reproducing their basic features requires different state connectivity: whether the desensitized state is accessed from the resting or active state. It is surprising that a property as fundamental as state connectivity would differ within the same family. So, we explore the possibility that the connectivity is the same, but corresponding states differ in function: Analogous states on the free energy landscape have similar structure, but differ in ion conductivity, free energies, and agonist binding affinities. This hypothesis is tested using a kinetic model in which agonist and anesthetics modulate the receptor free energy landscape by adsorbing to the membrane in which the receptor is embedded. It was previously shown that even with only three states, the complex behavior observed for GABAAR is reproduced, including its response to anesthetics. It is demonstrated here that this hypothesis accounts for an important difference between inhibitory and excitatory receptors: their opposite responses to inhalation anesthetics.

A chemical shift encoding (CSE) approach for spectral selection in fluorine-19 MRI.

PURPOSE: To develop a chemical shift encoding (CSE) approach for fluorine-19 MRI of perfluorocarbons in the presence of multiple known fluorinated chemical species. THEORY AND METHODS: A multi-echo CSE technique is applied for spectral separation of the perfluorocarbon perfluoro-15-crown-5-ether (PFCE) and isoflurane (ISO) based on their chemical shifts at 4.7 T. Cramér-Rao lower bound analysis is used to identify echo combinations with optimal signal-to-noise performance. Signal contributions are fit with a multispectral fluorine signal model using a non-linear least squares estimation reconstruction directly from k-space data. This CSE approach is tested in fluorine-19 phantoms and in a mouse with a 2D and 3D spoiled gradient-echo acquisition using multiple echo times determined from Cramér-Rao lower bound analysis. RESULTS: Cramér-Rao lower bound analysis for PFCE and ISO separation shows signal-to-noise performance is maximized with a 0.33 ms echo separation. A linear behavior (R2 = 0.987) between PFCE signal and known relative PFCE volume is observed in CSE reconstructed images using a mixed PFCE/ISO phantom. Effective spatial and spectral separation of PFCE and ISO is shown in phantoms and in vivo. CONCLUSION: Feasibility of a gradient-echo CSE acquisition and image reconstruction approach with optimized noise performance is demonstrated through fluorine-19 MRI of PFCE with effective removal of ISO signal contributions. Magn Reson Med 79:2183-2189, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Important role of calcium chloride in preventing carbon monoxide generation during desflurane degradation with alkali hydroxide-free carbon dioxide absorbents.

We investigated whether calcium chloride (CaCl2), a supplementary additive in carbon dioxide (CO2) absorbents, could affect carbon monoxide (CO) production caused by desflurane degradation, using a Japanese alkali-free CO2 absorbent Yabashi Lime®-f (YL-f), its CaCl2-free and 1% CaCl2-added derivatives, and other commercially available alkali-free absorbents with or without CaCl2. The reaction between 1 L of desflurane gas (3-10%) and 20 g of desiccated specimen was performed in an artificial closed-circuit anesthesia system for 3 min at 20 or 40 °C. The CO concentration was measured using a gas chromatograph equipped with a semiconductor sensor detector. The systems were validated by detecting dose-dependent CO production with an alkali hydroxide-containing CO2 absorbent, Sodasorb®. Compared with YL-f, the CaCl2-free derivative caused the production of significantly more CO, while the 1% CaCl2-added derivative caused the production of a comparable amount of CO. These phenomena were confirmed using commercially available absorbents AMSORB® PLUS, an alkali-free absorbent with CaCl2, and LoFloSorb™, an alkali-free absorbent without CaCl2. These results suggest that CaCl2 plays an important role in preventing CO generation caused by desflurane degradation with alkali hydroxide-free CO2 absorbents like YL-f.

Tunable Porous Coordination Polymers for the Capture, Recovery and Storage of Inhalation Anesthetics.

The uptake of inhalation anesthetics by three topologically identical frameworks is described. The 3D network materials, which possess square channels of different dimensions, are formed from the relatively simple combination of ZnII centres and dianionic ligands that contain a phenolate and a carboxylate group at opposite ends. All three framework materials are able to adsorb N2 O, Xe and isoflurane. Whereas the framework with the widest channels is able to adsorb large quantities of the various guests from the gas phase, the frameworks with the narrower channels have superior binding enthalpies and exhibit higher levels of retention. The use of ligands in which substituents are bound to the aromatic rings of the bridging ligands offers great scope for tuning the adsorption properties of the framework materials.

A membrane-embedded pathway delivers general anesthetics to two interacting binding sites in the Gloeobacter violaceus ion channel.

General anesthetics exert their effects on the central nervous system by acting on ion channels, most notably pentameric ligand-gated ion channels. Although numerous studies have focused on pentameric ligand-gated ion channels, the details of anesthetic binding and channel modulation are still debated. A better understanding of the anesthetic mechanism of action is necessary for the development of safer and more efficacious drugs. Herein, we present a computational study identifying two anesthetic binding sites in the transmembrane domain of the Gloeobacter violaceus ligand-gated ion channel (GLIC) channel, characterize the putative binding pathway, and observe structural changes associated with channel function. Molecular simulations of desflurane reveal a binding pathway to GLIC via a membrane-embedded tunnel using an intrasubunit protein lumen as the conduit, an observation that explains the Meyer-Overton hypothesis, or why the lipophilicity of an anesthetic and its potency are generally proportional. Moreover, employing high concentrations of ligand led to the identification of a second transmembrane site (TM2) that inhibits dissociation of anesthetic from the TM1 site and is consistent with the high concentrations of anesthetics required to achieve clinical effects. Finally, asymmetric binding patterns of anesthetic to the channel were found to promote an iris-like conformational change that constricts and dehydrates the ion pore, creating a 13.5 kcal/mol barrier to ion translocation. Together with previous studies, the simulations presented herein demonstrate a novel anesthetic binding site in GLIC that is accessed through a membrane-embedded tunnel and interacts with a previously known site, resulting in conformational changes that produce a non-conductive state of the channel.

Vapor Pressures of Anesthetic Agents at Temperatures Below 0°C and a Novel Anesthetic Delivery Device.

At room temperature, the vapor pressures of desflurane, isoflurane, and sevoflurane are well above the clinically useful range. We hypothesized that therapeutic concentrations of these agents could be achieved at temperatures below 0°C, but the vapor pressure-temperature relationship is unknown below 0. Second, we hypothesized that this relationship could be exploited to deliver therapeutic-range concentrations of anesthetic vapor. We therefore set out to determine the low temperature-vapor pressure relationships of each anesthetic agent, thereby identifying the saturated vapor concentration of each agent at any temperature below 0°C. To test our hypothesis, we measured the saturated vapor concentration at 1 atm of pressure for temperatures between -60 and 0°C, thus developing an empiric relationship for each agent. There was consistency in repeated experiments for all 3 agents. To test the empiric data, we constructed a digitally controlled thermoelectric anesthetic vaporizer, characterized the device, and used it to deliver anesthetic vapor to laboratory mice. We report, for the first time, the temperature-vapor pressure relationship at temperatures below 0°C for desflurane, isoflurane, and sevoflurane as well as the TMAC of these agents: the temperature at which the vapor pressure is equal to the minimum alveolar concentration. We describe the construction and limited validation of an anesthetic vaporizer prototype on the basis of this principle. We conclude that clinically relevant concentrations of volatile anesthetics may be achieved at low temperatures.