n-Dodecyl β-D-maltoside specifically competes with general anesthetics for anesthetic binding sites

We recently demonstrated that the anionic detergent sodium dodecyl sulfate (SDS) specifically interacts with the anesthetic binding site in horse spleen apoferritin, a soluble protein which models anesthetic binding sites in receptors. This raises the possibility of other detergents similarly interacting with and occluding such sites from anesthetics, thereby preventing the proper identification of novel anesthetic binding sites. n-Dodecyl β-D-maltoside (DDM) is a non-ionic detergent commonly used during protein-anesthetic studies because of its mild and non-denaturing properties. In this study, we demonstrate that SDS and DDM occupy anesthetic binding sites in the model proteins human serum albumin (HSA) and horse spleen apoferritin and thereby inhibit the binding of the general anesthetics propofol and isoflurane. DDM specifically interacts with HSA (K_d?=?40?μM) with a lower affinity than SDS (K_d?=?2?μM). DDM exerts all these effects while not perturbing the native structures of either model protein. Computational calculations corroborated the experimental results by demonstrating that the binding sites for DDM and both anesthetics on the model proteins overlapped. Collectively, our results indicate that DDM and SDS specifically interact with anesthetic binding sites and may thus prevent the identification of novel anesthetic sites. Special precaution should be taken when undertaking and interpreting results from protein-anesthetic investigations utilizing detergents like SDS and DDM.     

Liposomal-benzocaine gel formulation: correlation between in vitro assays and in vivo topical anesthesia in volunteers

The aim of the present study was to characterize a liposome-based benzocaine (BZC) formulation designed for topical use on the oral mucosa and to evaluate its in vitro retention and permeation using the Franz-type diffusion cells through pig esophagus mucosa. To predict the effectiveness of new designed formulations during preclinical studies, a correlation between in vitro assays and in vivo efficacy was performed. Liposomal BZC was characterized in terms of membrane/water partition coefficient, encapsulation efficiency, size, polydispersity, zeta potential, and morphology. Liposomal BZC (BL10) was incorporated into gel formulation and its performances were compared to plain BZC gel (B10) and the commercially available BZC gel (B20). BL10 and B10 presented higher flux and retention on pig esophagus mucosa with a shorter lag time, when compared to B20. BZC flux was strongly correlated with in vivo anesthetic efficacy, but not with topical anesthesia duration. The retention studies did not correlate with any of the in vivo efficacy parameters. Thus, in vitro permeation study can be useful to predict anesthetic efficacy during preclinical tests, because a correlation between flux and anesthetic efficacy was observed. Therefore, in vitro assays, followed by in vivo efficacy, are necessary to confirm anesthetic performance.     

Genetic effects on an anesthetic‐sensitive pathway in the brain of drosophila

General anesthetics are known to inhibit the electrically induced escape response of the fruitfly through action within the brain. We examined this response and its sensitivity to anesthetics in several mutants that cause significant disruption of the mushroom body and other structures of the central brain in adult flies. Because we show here that anesthesia sensitivity is influenced by genetic background, we have used a set of congenic mutant lines. Sensitivity to halothane is normal in most of these lines, indicating that the anesthetic target is unaffected by the gross status of the central brain. Thus, for the escape response, anesthetic sensitivity is not a global feature but reflects action at a localized target. Only the mushroom body defect (mud) line showed an increased sensitivity of the escape response to halothane. Sensitivity to two other anesthetics is also perturbed in this line, albeit less dramatically so. The behavior of mud/+ heterozygotes and the comparison of brain anatomy among all the mutant lines imply that the effect of the mud mutation on anesthesia is not via gross alteration of central brain structures. The possibility that an adventitious mutation in the mud line is responsible for the effects on anesthesia is disfavored by the behavior of a heterozygote between two mud alleles. Although we do not yet know whether the mud gene encodes an anesthetic target or influences the functioning of an anesthetic‐sensitive neuron in this pathway, our work indicates that this gene regulates the effects of halothane on a circumscribed pathway. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 69–78, 2000     

Reversal by pressure of seed germination promoted by anesthetics

Germination of Panicum capillare L. caryopses induced by solutions of ethanol and ethyl ether was prevented by application of pressure >1 MPa during the period of exposure to the anesthetic. This effect of pressure indicates that germination is correlated with expansion at a site of anesthetic action in a cell membrane. The effects of several other anesthetics were measured on germination of P. capillare seeds. Ethanol, chloroform, and ethyl ether had the highest activity. Methanol and isopropanol were inactive. The effective compounds are thought to distribute preferentially to lipid-solution interfaces in cell membranes of the seeds.     

In vivo and analytical studies of forces and moments in equine long bones

A study to evaluate the in vivo forces acting on equine long bones is described. Results for the maximum axial forces and bending moments are presented for the radius, metacarpus, tibia, and metatarsus. The forces and bending moments are obtained by combining in vivo strain gage readings and a mathematical model for the mechanics of the bones. Comparisons of equine activities including standing, walking, trotting, and getting up after the anesthetic indicate that one activity does not always produce the highest loads in all of the bones examined. However, for the cases considered here, recovery from the anesthetic produced the largest compressive forces in the metacarpus and the tibia. Also, large tensile forces were found during the foot-up positions during standing and trotting.     

Volatile general anesthetics reveal a neurobiological role for the white and brown genes of Drosophila melanogaster

Molecular and cellular evidence argues that a heterodimer between two ABC transporters, the White protein and the Brown protein, is responsible for pumping guanine into pigment‐synthesizing cells of the fruit fly, Drosophila melanogaster. Previous studies have not detected White or Brown outside pigment‐synthesizing cells nor have behavioral effects of null mutants been reported, other than those that are visually dependent. Nevertheless, we show here that exposure to the volatile general anesthetic (VGA) enflurane reveals a difference in neuromuscular performance between wild‐type flies and those that carry a null allele in either the white or brown gene. Specifically, in a test of climbing ability, w¹¹¹⁸ or bw¹ flies are much less affected by enflurane than are congenic controls. Altered anesthetic sensitivity is still observed when visual cues are reduced or eliminated, arguing that white and brown contribute to neural function outside the eye. This hypothesis is supported by the detection of white message in heads of flies that are genetically altered so as to lack pigment‐producing cells. The w¹¹¹⁸ or bw¹ mutations also alter the response to a second VGA, halothane, albeit somewhat differently. Under some conditions, the combination of w¹¹¹⁸ with another mutation that affects anesthesia leads to a drastically altered phenotype. We consider several ways by which diminished transport of guanine could influence neural function and anesthetic sensitivity. Published 2001 John Wiley & Sons, Inc. J Neurobiol 49: 339–349, 2001     

Essential oil of Lippia alba as a sedative and anesthetic for the sea urchin Echinometra lucunter (Linnaeus, 1758)

The sea urchin, Echinometra lucunter, is consumed in restaurants in Europe and Asia for high prices. This study evaluated the use of the essential oil of Lippia alba (EOL) as a sedative and anesthetic in this sea urchin. Different EOL concentrations were tested (50, 100 and 150 μL L^?1), in addition to a group that received ethanol and a control group. After the anesthesia and recovery from the different treatments, the coelomic fluid, gonads and intestine were collected for the analysis of oxidative stress parameters. The highest concentration tested (150 μL L^?1) was determined to be the optimal concentration for anesthesia. Results suggest that EOL improved the response to oxidative stress, since a lower level of thiobarbituric acid-reactive substances and greater superoxide dismutase and catalase activities were observed in the coelomic fluid and E. lucunter gonads, making the EOL a promising anesthetic for sea urchins.     

Chronic exposure to inhaled anesthetics increases cholesterol content in Acholeplasma laidlawii

Acholeplasma laidlawii cells were grown in cholesterol-enriched medium and exposed continuously to either air (control), 4.0 vol.% halothane in air at 1 atm pressure (4% atm halothane), or 80% cyclopropane in oxygen for 24 h at 37°C. Cells grown in the presence of 4% atm halothane or 80% cyclopropane had approximately twice as much membrane cholesterol content/mg protein as the control cells. Cells grown in an anesthetic environment also tended to have a higher membrane cholesterol/phospholipid molar ratio compared to control cells. Membranes isolated from halothane-exposed cells grown in a cholesterol-enriched medium were more ordered at 37°C (measurements were made with no anesthetic present) than membranes from control cells grown in an identically enriched medium. This difference in membrane physical state between control and anesthetic-exposed cells decreased as the temperature decreased, and disappeared at approx. 23°C. Continuous exposure of A. laidlawii to 4% atm halothane or 80% cyclopropane for 24 h did not markedly affect membrane fatty acid composition, either in cells grown on an unsupplemented medium or in cells grown in a medium enriched in myristic, palmitic or stearic acids. These results further support the hypothesis that an increased membrane cholesterol content may play a role in the tolerance or dependence that develops after chronic exposure to anesthetic agents.     

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.     

Comparison of Isoflurane and Carbon Dioxide Anesthesia in Chilean Rose Tarantulas (Grammostola rosea)

This study investigated the use of two anesthetic agents, isoflurane and carbon dioxide, in Chilean rose tarantulas (Grammostola rosea). We compared the onset, duration of anesthesia, and recovery time with both gases, and made observations regarding the effects of the anesthetic protocols. Subjectively, episodes for the isoflurane animals were uneventful. The spiders were calm throughout and did not respond adversely to gas exposure. Conversely, animals anesthetized with carbon dioxide experienced violent inductions and recoveries; the tarantulas appeared agitated when the carbon dioxide flow began. Seizure‐like activity and defecation would frequently be noted prior to induction with carbon dioxide. Neither isoflurane nor carbon dioxide seemed to have any clinically apparent short‐ or long‐term impact. The animals were all normal for at least 1‐year postexperiment. Future studies should focus on defining the impact, if any, that these anesthetic agents have on the health of invertebrate species. Zoo Biol. 32:101‐103, 2013. © 2012 Wiley Periodicals, Inc.     

Taking the sting out of darting: Risks,restraint drugs and procedures for the chemical restraint of Southern Hemisphere otariids

The need to manage otariid populations has necessitated the development of a wide range of capture methods. Chemical restraint by remote drug delivery (i.e., darting) is a highly selective method that can be used to facilitate otariid capture in a range of scenarios, when other methods may be impracticable. However, the risks associated with darting otariids are not widely known and guidelines necessary to promote and refine best practice do not exist. We review the risks associated with darting and in light of our findings, develop darting guidelines to help practitioners assess and minimize risks during capture, anesthesia and recovery. Published studies reveal that mortalities associated with darting predominantly result from complications during anesthetic maintenance (e.g., prolonged respiratory depression, apnea, or hyperthermia), rather than from complications during capture or recovery. In addition to monitoring vital signs and proper intervention, the risk of irreversible complications during anesthesia can be reduced by administering drug doses that are sufficient to enable the capture and masking of animals, after which anesthetic depth can be regulated using gas anesthesia.     

Transfection of liver in vivo by biolistic particle delivery

We describe the delivery of reporter gene constructs to rat liver through the use of the Helios Gene Gun system. The effectiveness of this transfection method is illustrated by describing its use for determining in vivo the role of a DNA element that regulates cytochrome P450 2B1 (CYP2B1) gene expression in response to xenobiotics. DNA was delivered to the liver of an anesthetized animal via DNA-coated gold microcarriers. The highest level of reporter gene expression was obtained about six hours posttransfection; however, at this time endogenous CYP2B1 mRNA is transiently induced by the anesthetic treatment. The optimal time for investigating expression of a reporter gene under the control of CYP2B1 regulatory elements was 24 h after transfection, by which time the inductive effect of the anesthetic had ceased. Reporter gene expression subsequently declined rapidly to a low level by 48 h. In the transfected liver the heterologous SV40 promoter was about eight-fold stronger than the minimal CYP2B1 promoter. However, when attached to the phenobarbital response element both promoters give the same fold-induction of reporter activity in response to phenobarbital.     

Isoflurane alters the structure and dynamics of GLIC

Pentameric ligand-gated ion channels are targets of general anesthetics. Although the search for discrete anesthetic binding sites has achieved some degree of success, little is known regarding how anesthetics work after the events of binding. Using the crystal structures of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), which is sensitive to a variety of general anesthetics, we performed multiple molecular dynamics simulations in the presence and absence of the general anesthetic isoflurane. Isoflurane bound to several locations within GLIC, including the transmembrane pocket identified crystallographically, the extracellular (EC) domain, and the interface of the EC and transmembrane domains. Isoflurane also entered the channel after the pore was dehydrated in one of the simulations. Isoflurane disrupted the quaternary structure of GLIC, as evidenced in a striking association between the binding and breakage of intersubunit salt bridges in the EC domain. The pore-lining helix experienced lateral and inward radial tilting motion that contributed to the channel closure. Isoflurane binding introduced strong anticorrelated motions between different subunits of GLIC. The demonstrated structural and dynamical modulations by isoflurane aid in the understanding of the underlying mechanism of anesthetic inhibition of GLIC and possibly other homologous pentameric ligand-gated ion channels.     

The effect in vitro of volatile anesthetics on the activity of cholinesterases.

Because the mechanism of anesthesia is unknown, the relationship between anesthetics and enzymes essential to brain function may be an important one. Therefore, the effect of 8 volatile anesthetics on the enzymatic activity of solubilized, purified dog brain and human erythrocyte acetylcholinesterase (AChE) and human serum cholinesterase (ChE) was studied in vitro. Serum ChE was found to be insensitive to saturated solutions of all the anesthetics studied. However, brain and erythrocyte AChE were reversibly inhibited in a dose-dependent manner by all 8 anesthetics in concentrations exceeding those used in clinical practice. Kinetic analysis revealed a mixed (competitive, non-competitive) type of inhibition with the exception of the ether-crythrocyte AChE interaction which was characterized by competitive inhibition. Ether and methoxyflurane were found to depress the AChE activity the most and isoflurane and enflurane the least. The concentrations of anesthetic in the gas phase necessary for 50% inhibition of erythrocyte AChE activity (I₅₀) were calculated for 5 anesthetics and found to correlate with their water-gas partition coefficients. These data suggest that the effect in vitro of volatile anesthetics on the catalytic activity of cholinesterases is a variable one and may be unrelated to anesthetic potency in vivo. The implications of these data concerning anesthetic-active site interactions are discussed.     

Relationship between gastrointestinal transit time and anesthetic fasting protocols in the captive chimpanzee, Pan troglodytes

Background Lengthy social separation and prolonged fasting time contribute to increased risks associated with anesthesia in captive primates. This study is an initial attempt to identify a safe pre‐anesthetic fasting procedure by identifying gastric emptying time (GET) and gastrointestinal transit time (GTT) of captive chimpanzees, Pan troglodytes. Methods Seven adult chimpanzees at the North Carolina Zoo immobilized for annual physical examinations were fed barium‐impregnated polyethylene spheres to measure GET. Eleven animals were individually fed a color dye marker and fecal passage was observed to determine GTT. Results Gastric emptying time (GET) was approximated to be >3 hours but <16 hours. The mean GTT was 16.5 hours. Conclusions This study indicates that a fasting time of 3 hours would allow for complete gastric emptying and could potentially replace the current overnight fast (≥16 hour) to help minimize complications associated with pre‐anesthetic fasting in captive primates.     

Effect of preparation technique on the properties and in vivo efficacy of benzocaine-loaded ethosomes

This study aimed to investigate the influence of the preparation conditions on the performance of an ethosomal formulation for topical delivery of the local anesthetic agent, benzocaine (BZC). Ethosomes were prepared with different techniques, such as thin-layer evaporation, freezing and thawing, reverse-phase evaporation, extrusion and sonication, obtaining, respectively, multilayer vesicles (MLVs), frozen and thawed MLV (FATMLV), large unilamellar vesicles (LUVs), and small unilamellar vesicles (SUVs). The obtained vesicles were characterized for morphology, size, zeta potential, and entrapment efficiency (EE%), and their stability was monitored during storage at 4°C. In vitro permeation properties from gels incorporating drug ethosomal dispersions were evaluated in vitro by using artificial lipophilic membranes, while their anesthetic effect was determined in vivo on rabbits. The results suggested that the vesicle preparation method plays an important role in affecting the properties and effectiveness of ethosomal formulations. MLVs and LUVs exhibited higher drug EE% and better stability than FATMLV and SUV vesicles. The In vitro drug permeation rate was directly related to the vesicle EE% and varied in the order MLV>LUV≈FATMLV>SUV. The therapeutic efficacy of BZC ethosomal formulations was significantly improved with respect to the corresponding BZC solution. The best results, in terms of enhanced intensity of anesthetic effect, were given by formulations containing MLVs and LUVs, and the order of effectiveness was MLV≈LUV>FATMLV≈SUV, rather similar to that found in permeation studies. On the contrary, unexpectedly, the effectiveness order in increasing the duration of drug action was SUV≥MLV>LUV≈FATMLV. The highest efficacy of SUVs was probably due to the more intimate contact with the epithelium due to their greatest surface area, which allowed the longest extension of drug therapeutic action. The overall results suggest that a suitably developed ethosomal formulation of BZC can be of actual value for improving its clinical effectiveness in topical anesthesia.     

Effects of nitrous oxide on mitochondrial and cell respiration and growth in Distichlis spicata suspension cultures

The reversible inhibition of respiratory activity could provide a novel approach to the preservation of traditionally hard to store plant germplasm such as clonal materials and recalcitrant seed. The gaseous anesthetic nitrous oxide caused a reversible, dose-dependent, partial inhibition of dioxygen utilization in mitochondrial particles isolated from cell suspension cultures of the salt-tolerant marsh grass Distichlis spicata, with maximal inhibition of 33% after 30 minutes exposure to an atmosphere of 80% nitrous oxide plus 20% oxygen. Respiration of whole cells required longer time to be affected by the anesthetic, and was reversibly inhibited an average of 19% when measured using a differential respirometer. Exposure to 80% nitrous oxide plus 20% oxygen for up to 10 days caused no measurable effect on cell growth.Abbreviations PCV packed cell volume - EDTA sodium ethylenediaminetetraacetic acid - BSA bovine serum albumin - MOPS 3(N-morpholino) propanesulfonic acid - TMPD N,N,N',N'-tetramethyl-p-phenylene diamine - STP standard temperature and pressure     

New volatile anesthetic,desflurane, reduces vitamin E level in blood of operative patients via oxidative stress

It has been well known that some volatile anesthetic agents produce oxidative stress. Desflurane as a new volatile agent might have limited oxidative toxic effect because it is relatively a new short‐acting anesthetic characterized by a short duration of action and a quick postanesthetic recovery. We investigated effect of desflurane on serum glutathione peroxidase (GSH‐Px), lipid peroxidation (LP), vitamin E, and erythrocyte superoxide dismutase (SOD) values in patients. Fifteen adult patients are scheduled for elective surgery, ASA I or II physical status. Tidal volume and ventilation frequency were kept unchanged during the operation. Baseline values in venous blood samples were preoperatively taken and blood was also taken postoperatively at the 1st and the 12th hours of desflurane exposure. LP levels were significantly (p < 0.05) higher postoperatively at 1st hour than in preoperative values while α‐tocopherol concentration was significantly (p < 0.001) lower in postoperative period at 1st hour than in preoperative period. Erythrocyte SOD and serum GSH‐Px activities did not differ between pre‐ and postoperative periods. In conclusion, we observed that desflurane produced oxidative stress by decreasing α‐tocopherol levels. Use of vitamin E may be possible to reduce the oxidative effect of desflurane. Copyright © 2010 John Wiley & Sons, Ltd.     

Evaluation of anesthetic protocol for the collection of semen from captive collared peccaries (Tayassu tajacu) by electroejaculation

The objective of this study is to verify and compare the effects of acepromazine–tiletamine–zolazepam and propofol used in anesthetic protocols for semen collection by electroejaculation from captive collared peccaries. Ten sexually mature animals were physically restrained and anesthetized by either intravenous administration of tiletamine–zolazepam (2 mg/kg) after acepromazine premedication, or a propofol dose of 5 mg/kg. The onset of anesthetic recovery was determined by the animals regaining consciousness and attempting to stand. Semen was collected by electroejaculation and evaluated for volume, pH, sperm concentration, progressive motility, morphology, percentage of live cells and functional membrane integrity. Six anesthetized animals with the acepromazine–tiletamine–zolazepam protocol showed erection, but semen could be collected in only four (40%) attempts. Of the animals anesthetized using propofol, nine showed erection, and the ejaculates were collected in eight (80%) attempts. Furthermore, propofol afforded rapid recovery of animals, and ejaculates with enhanced sperm motility and functional membrane integrity as compared with those collected by the other protocol (P < 0.05). In conclusion, use of propofol for anesthetic restraint of collared peccaries enhanced collection of semen by electroejaculation.     

Evaluation of three immobilization combinations in the capybara (Hydrochoerus hydrochaeris)

Capybaras (Hydrochoerus hydrochaeris) are the world's largest rodent. Owing to its uniqueness, 50 AZA institutions in North America display this species. As shown by a survey, no standard anesthetic protocol has been developed for this species. As a part of an ongoing behavioral study in Venezuela, capybaras were surgically implanted with radio transmitters. Animals were randomly assigned to one of the three immobilization protocols: (1) Tiletamine HCl/Zolazepam HCl, (2) Tiletamine HCl/Zolazepam HCl/Medetomidine HCl, and (3) Tiletamine HCl/Zolazepam HCl/Medetomidine HCl/Butorphanol tartrate. The protocol recommended for minimally invasive procedures when inhalant anesthetics are unavailable is a combination of Tiletamine HCl/Zolazepam HCl/Medetomidine HCl/Butorphanol tartrate. This is based on ease of administration, volume, onset of action, depth of anesthetic achieved, reversibility, safety, and costs. Zoo Biol 29:59–67, 2010. © 2009 Wiley‐Liss, Inc.