A vertebrate model to reveal neural substrates underlying the transitions between conscious and unconscious states.

The field of neuropharmacology has not yet achieved a full understanding of how the brain transitions between states of consciousness and drug-induced unconsciousness, or anesthesia. Many small molecules are used to alter human consciousness, but the repertoire of underlying molecular targets, and thereby the genes, are incompletely understood. Here we describe a robust larval zebrafish model of anesthetic action, from sedation to general anesthesia. We use loss of movement under three different conditions, spontaneous movement, electrical stimulation or a tap, as a surrogate for sedation and general anesthesia, respectively. Using these behavioral patterns, we find that larval zebrafish respond to inhalational and IV anesthetics at concentrations similar to mammals. Additionally, known sedative drugs cause loss of spontaneous larval movement but not to the tap response. This robust, highly tractable vertebrate model can be used in the detection of genes and neural substrates involved in the transition from consciousness to unconsciousness.

Development and validation of brain target controlled infusion of propofol in mice.

Mechanisms through which anesthetics disrupt neuronal activity are incompletely understood. In order to study anesthetic mechanisms in the intact brain, tight control over anesthetic pharmacology in a genetically and neurophysiologically accessible animal model is essential. Here, we developed a pharmacokinetic model that quantitatively describes propofol distribution into and elimination out of the brain. To develop the model, we used jugular venous catheters to infuse propofol in mice and measured propofol concentration in serial timed brain and blood samples using high performance liquid chromatography (HPLC). We then used adaptive fitting procedures to find parameters of a three compartment pharmacokinetic model such that all measurements collected in the blood and in the brain across different infusion schemes are fit by a single model. The purpose of the model was to develop target controlled infusion (TCI) capable of maintaining constant brain propofol concentration at the desired level. We validated the model for two different targeted concentrations in independent cohorts of experiments not used for model fitting. The predictions made by the model were unbiased, and the measured brain concentration was indistinguishable from the targeted concentration. We also verified that at the targeted concentration, state of anesthesia evidenced by slowing of the electroencephalogram and behavioral unresponsiveness was attained. Thus, we developed a useful tool for performing experiments necessitating use of anesthetics and for the investigation of mechanisms of action of propofol in mice.

Cardiopulmonary bypass, hemolysis, and nitroprusside-induced cyanide production.

BACKGROUND: Cyanide toxicity is a complication of sodium nitroprusside administration. Cardiac surgery may increase the risk of cyanide toxicity, because hemolysis during cardiopulmonary bypass (CPB) may catalyze the release of free cyanide from sodium nitroprusside. METHODS: We obtained serial blood specimens from 25 cardiac surgical patients during CPB. Plasma specimens were analyzed for free hemoglobin concentration and ability to generate free cyanide anion upon exposure to sodium nitroprusside. RESULTS: Hemolysis based on plasma-free hemoglobin concentration increased over time during CPB at an average rate of 0.27 mg x dL(-1) x min(-1) (P < 0.001). The concentration of free cyanide generated by the addition of sodium nitroprusside to the plasma samples was directly related to the plasma-free hemoglobin concentration (P < 0.001). CONCLUSION: CPB-associated hemolysis and free hemoglobin release accelerated the immediate release of free cyanide from sodium nitroprusside. These in vitro findings suggest that cardiac surgical patients may be at increased risk of cyanide toxicity in response to the perioperative administration of sodium nitroprusside.

Measurement of resiniferatoxin in serum samples by high-performance liquid chromatography.

A novel and simple method of extraction, separation, identification and quantification of resiniferatoxin (RTX) in serum samples is reported. Human serum and whole blood were treated with acetonitrile to denature proteins, such as orosomucoid, and the soluble fraction was passed through a reversed-phase C18 cartridge. RTX eluted from the cartridge was quantified by high-performance liquid chromatography (HPLC) using a reversed-phase C18 column. Reproducible recovery of RTX and tinyatoxin, an internal standard, from serum was achieved. Isocratic elution with 62% acetonitrile provided a suitable retention time without interfering peaks eluting near the analyte. Therefore, the procedure described provides a useful assay for determination of serum RTX pharmacokinetic parameters.

Chromatographic approach for determining the relative membrane permeability of drugs.

With the aid of the experimental dependence of the theoretical plate height (H) on the flow-rate (U), values of diffusion coefficients as the permeation rate, of the compounds in a polymeric stationary phase were calculated from solute mass transfer. This approach is proposed for modeling the relative diffusion rate of a drug within the membrane. After the successful separation of opioid compounds using a C(18) derivatized polystyrene-divinylbenzene polymer HPLC column, the slopes of H-U plots increase quantitatively in the order of meperidine

Measurement of resiniferatoxin in cerebrospinal fluid by high-performance liquid chromatography.

A sensitive and simple high-performance liquid chromatographic (HPLC) assay was developed for the quantification of resiniferatoxin (RTX) in canine cerebrospinal fluid (CSF). A reversed-phase C(18) column and acetonitrile in 0.02 M NaH(2)PO(4) as mobile phase provided satisfactory resolution for RTX analysis. Direct HPLC analysis of the CSF samples without sample extraction or preparation improves the accuracy and detection limits of this assay. This assay was applied to measure CSF RTX content to test this method for research and clinical applications related to studies examining its analgesia effects.

Low-affinity analytical chromatography for measuring inhaled anesthetic binding to isolated proteins.

The direct measure of volatile anesthetic binding to protein is complicated by weak affinity and therefore rapid kinetics. Consequently, several puted targets for these clinically important drugs have only functional data to support a direct mode of action. While several methods for measuring some aspects of binding are available, all have significant limitations. We introduce the use of analytical chromatography for the purpose of directly measuring volatile anesthetic binding to protein, and show that it can provide estimates of both affinity and stoichiometry for proteins that can be obtained in fairly high purity and mass. Using this approach we characterize halothane binding to serum albumin as low affinity and multisite, and to myoglobin or cytochrome C as strictly nonspecific. This approach will be useful in directly characterizing equilibrium, solution binding to isolated proteins in preparation for more time-consuming methods with structural resolution.