Selective breeding of mice strains with different sensitivity to isoflurane

Background The mechanisms of action of volatile anesthetics are still unknown. Recently, the use of genetics as ameans to investigate anesthetic action has increased in scale. However, only limited forward genetic approach studieswere performed in mammals, especially with volatile anesthetics as the selection agent. In the present study, a selectivebreeding process was designed to produce strains of mice with different sensitivity to isoflurane.Methods One hundred and sixty male and female virgin outbred ICR/CD-1 mice at 65-70 days of age were selected asoriginal generation, and the median effective dose (ED) of inhaled isoflurane were measured by probit analysis with theloss of righting reflex as the endpoint of anesthesia. The most sensitive males and females were selected and mated oneanother randomly, as with the most resistant males and females. Thus two branches of mice （sensitive and resistant toisoflurane） were created and allowed to produce the next generation. At 65-70 days of age, screening experiment wasperformed in offspring, by selecting the most sensitive mice in sensitive branch and the most resistant mice in resistantbranch. Selected males and females within each branch were mated one another randomly to produce the followinggeneration. The same procedure was performed in the offspring. The process of screening and breeding was repeatedfor 8 generations, and then strains were conserved by mating the offspring one another randomly within each branch for3 generations. Each pair of mice was allowed to produce the second litters as a backup, and isoflurane EDwasmeasured in mice from the second litters.Results Isoflurane righting reflex EDs (95% confidence limit （CL）) in original mice were 0.65% （0.58%-0.72%） infemales and 0.63% （0.56%-0.69%） in males. After the 4th generation, isoflurane EDs in resistant branch weresignificantly higher than those in sensitive branch （P <0.05）, for both in females and males. In the 11th generation,isoflurane EDin the two branches differed by 32% in females and 36% in males.Conclusions After 8 generations of selective breeding and 3 generations of strain conservation, two strains of micewith high and low sensitivity to isoflurane were developed. The separation of inhaled anesthetic requirement in parentscould be transferred to the offspring in mice.