Victoria J. Simpson

Identification of a genetic region in mice that specifies sensitivity to propofol.

Background Long‐sleep (LS) and short‐sleep (SS) mice, initially selected for differential sensitivity to ethanol, also exhibit differential sensitivity to propofol. By interbreeding LS and SS mice to obtain progeny whose chromosomes are a patchwork of the LS and SS chromosomes, the authors determined whether differential propofol sensitivity cosegregates with any particular chromosomal region(s). Such cosegregation is the essence of genetic linkage mapping and a first step toward isolating a gene that can modulate propofol sensitivity in mammals. A gene underlying a quantitative trait such as anesthetic sensitivity is commonly called a quantitative trait locus (QTL). Methods The propofol dose was 20 mg/kg injected retroorbitally. Sensitivity was measured as the duration of the loss of righting reflex (LORR). The LORR and propofol brain levels at awakening were determined for 24 LSXSS recombinant‐inbred (RI) strains, derived by intercrossing LS and SS for two generations followed by > 20 generations of inbreeding. A genetic linkage between LORR and an albino mutation on chromosome 7 was investigated further using 164 second‐generation progeny (F2 s) from intercrossing inbred LS and inbred SS mice, similar to the LSXSS RIs except F2 s are not inbred. The linkage between propofol sensitivity and the albino locus also was investigated using additional genetic markers on chromosome 7. Statistical significance was assessed by interval mapping using a regression method for RIs and Mapmaker/QTL (Whitehead Institute, Cambridge, MA) for F2 s. Results Genetic mapping in the LSXSS RIs revealed a QTL tightly linked to the Tyr (albino) locus that accounts for nearly all of the genetic difference in propofol sensitivity between LS and SS mice. Analysis of propofol brain levels at awakening indicated that this QTL results from differential neurosensitivity. Mapping in F2 s confirmed the genetic linkage to Tyr. Mice (ISS c/c x C57BL/6 c2j /C) that differed only by an albino mutation at Tyr were not differentially sensitive to propofol. Conclusions A single QTL, called Lorp1, underlies most of the genetic difference in propofol neurosensitivity between LS and SS mice. Although this QTL is tightly linked to Tyr, propofol sensitivity is not modulated by albinism. For mapping this QTL, the LSXSS RIs proved to be an especially powerful resource, localizing the candidate‐gene region to a 99% confidence interval of only 2.5 centimorgans.

Propofol Produces Differences in Behavior but Not Chloride Channel Function Between Selected Lines of Mice

We report differential central nervous system (CNS) sensitivity to propofol between Long Sleep (LS) and Short Sleep (SS) mice, selectively bred for their differential CNS sensitivity to ethanol.Intravenous propofol requirements for loss of righting reflex, or sleep time, were measured to define the extent of this sensitivity. LS mice slept approximately two times longer than SS mice at equal doses. Awakening plasma and brain levels of the SS line were, respectively, two and three times that of the LS line (P < 0.0001). This suggests that the LS and SS sleep time difference is CNS mediated, and that propofol and ethanol may share common genes that determine anesthetic sensitivities. The ethanol effect may be at least partially mediated by gamma-aminobutyric acid-A (GABAA) receptor function. Propofol had no differential effect on GABAA receptor function, as measured by chloride flux in LS and SS brain microsac preparations. Either the GABAA receptor does not mediate propofol sleep time, or qualitative differences cannot be demonstrated using36 Cl- uptake in brain membranes. (Anesth Analg 1996;82:327-31)

GAS CHROMATOGRAPHY-MASS SPECTROMETRY ASSAY FOR DETERMINATION OF KETAMINE IN BRAIN

A sensitive and precise gas chromatography-mass spectrometry method with selected ion monitoring has been developed for determination of ketamine in the brain using chlorpheniramine as an internal standard. The assay is based on the acid extraction of brain homogenate with hexane and ethyl ether with subsequent alkaline ethyl ether extraction. The analytical procedure has a coefficient of variation of 3.0-5.3% and from 3.8 to 6.1% for extraction from water or spiked brain samples, respectively. The lowest detectable level of ketamine was 1 ng in any brain region. This level of detection was used to measure the ketamine concentrations in cerebellum, brain stem, midbrain, hypothalamus, and cortex of C57B1/6 mice at awakening following intraperitoneal injection of a hypnotic dose. The ketamine concentrations in mouse brain were in the range from 41.6 to 48.6 ng/mg of tissue.

Genetic Models in The Study of Anesthetic Drug Action

This chapter reviews the use of genetic models in the study of anesthetic drug action. Genetic model systems provide a novel approach to understanding mechanisms of anesthetic drug action. Many models have been derived using selection processes that emphasize differential drug sensitivity, producing animal lines that differ in their CNS drug response. Studies of vertebrate (rodent) and invertebrate (Drosophila, Caenorhabditis elegans) animal model systems are covered. The review discusses studies employing lines derived from spontaneous and induced mutagenic processes, selectively bred lines, and inbred lines possessing inherent differential drug sensitivities. The primary focus of included studies is the general anesthetic drugs that are commonly used in the clinical setting. These are drugs such as the inhalational agents (halothane, enflurane, isoflurane, nitrous oxide) and the intravenous induction agents (propofol and diazepam). Rodent lines with differential sensitivity to opiates are also discussed. Finally, an approach to identifying and isolating the genes that control anesthetic sensitivity is discussed in a section on mapping quantitative trait loci (QTL) in recombinant inbred lines.

Mapping quantitative trait loci mediating sensitivity to etomidate.

Long- and Short-Sleep (LS and SS) mice were selectively bred for differences in ethanol-induced loss of the righting reflex (LORR) and have been found to differ in LORR induced by various anesthetic agents. We used a two-stage mapping strategy to identify quantitative trait loci (QTLs) affecting duration of LORR caused by the general anesthetic etomidate and brain levels of etomidate (BEL) following regain of the righting reflex. Analysis of recombinant-inbred strains derived from a cross between LS and SS mice (LSXSS) yielded a heritability estimate of 0.23 for etomidate-induced LORR and identified one marker that showed suggestive linkage for a QTL, on mouse Chromosome (chr) 12. Mapping in an F2 population derived from a cross between inbred LS and SS (ILS and ISS) revealed a significant QTL for etomidate-induced LORR on Chr 12, and two significant QTLs mediating BEL on Chrs 6 and 12. Several QTLs showing suggestive linkage for etomidate-induced LORR and BEL were also identified in the F2 population. Brain levels of etomidate in the RI and F2 mice suggested that differences in LORR were due to differential central nervous system sensitivity, rather than differential etomidate metabolism. Interestingly, the region on Chr 7 has also been identified as a region influencing ethanol-induced LORR, suggesting the possibility of a common genetic mechanism mediating etomidate and ethanol sensitivity. These QTL regions need to be further narrowed before the testing of candidate genes is feasible.

Gas chromatographic–mass spectrometric determination of etomidate in mouse brain

A simple, rapid and reliable method was developed to determine the concentration of etomidate [ethyl-1-(1-phenylethyl)-1 H-imidazole-5-carboxylate] in mouse brain tissue by gas chromatography-mass spectrometry (GC-MS). Ethyl 5-amino-1-phenyl-4-pyrazolecarboxylate was used as internal standard (IS) and liquid-liquid extraction using ethyl ether as the solvent. The method demonstrated excellent recovery (93%) and a linear calibration range of 50-2500 ng/0.2 g. Intra-day accuracy and precision had an error and coefficient of variation of less than 8.7% and 4.2%, respectively. The limit of detection was 1 ng in mouse brain. Our results suggest that this new method is suitable for the quantitative analysis of etomidate in mouse brain tissue.

Genetic Models and Mapping Genes in the Study of Anaesthetic Action

The molecular action of anaesthetic agents is a problem well studied but not well understood. A novel approach to identifying molecular pathways involved in anaesthetic drug action involves isolating the genes mediating anaesthetic sensitivity in animal models. Several animal models have been derived using differential drug sensitivity as a screening phenotype. This method has produced both invertebrate (Drosophlla melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae) and vertebrate (rodent) animal lines that differ in their central nervous system (CNS) response to anaesthetic agents. Lines can be derived from spontaneous or induced mutagenic processes, or selective breeding schemes. Inbred and recombinant lines possessing inherent differential drug sensitivities also are available. All can be used in a method of gene mapping known as quantitative trait loci (QTL) mapping. Anaesthetic sensitivity is a quantitative measure and is likely influenced by multiple genes known as QTLs where each QTL corresponds to a single gene. In this review we describe several genetic models currently used in studying anaesthetic action. We focus primarily on a mouse model which has been used to identify a QTL mediating propofol neurosensitivity in a selectively bred mouse line known as Long Sleep (LS) and Short Sleep (SS) mice.