Fig 1.
C57BL/6J mice were anesthetized or euthanized using six different standard laboratory methods as described in the text. Skeletal muscle, serum, liver, heart and adipose tissue were then rapidly collected and frozen by immersion in liquid nitrogen. Tissue samples were pulverized under liquid nitrogen, then sonicated in extraction solvent. Samples were centrifuged and the supernatant was analyzed by LC-MS. Targeted and untargeted metabolomics were used to identify metabolites which differed in abundance between methods of anesthesia or euthanasia.
Fig 2.
Principal component analysis of untargeted metabolomics data from tissues collected using different methods of anesthesia.
Two-dimensional PCA score plots reveal separation in metabolite profiles induced by different methods of anesthesia and euthanasia in C57BL/6J mice. Tissues analyzed were a) skeletal muscle, b) heart, c) liver, d) white adipose and e) serum. Methods of anesthesia and euthanasia were: CD, cervical dislocation euthanasia (red); CO2, Carbon dioxide euthanasia (green); Iso-Cont, continuous isoflurane anesthesia (dark blue); Iso-OD, isoflurane overdose euthanasia (light blue); Ket, ketamine anesthesia (pink); Pent, pentobarbital anesthesia (orange). Ellipses represent the 95% confidence interval.
Fig 3.
Partial least squares discriminant analysis of untargeted metabolomics data from tissues collected using different methods of anesthesia.
Two-dimensional PLS-DA score plots reveal separation in metabolite profiles induced by different methods of anesthesia and euthanasia in C57BL/6J mice. Tissues analyzed were a) skeletal muscle, b) heart, c) liver, d) white adipose and e) serum. Methods of anesthesia and euthanasia were: CD, cervical dislocation euthanasia (red); CO2, Carbon dioxide euthanasia (green); Iso-Cont, continuous isoflurane anesthesia (dark blue); Iso-OD, isoflurane overdose euthanasia (light blue); Ket, ketamine anesthesia (pink); Pent, pentobarbital anesthesia (orange). Ellipses represent the 95% confidence interval.
Table 1.
Annotated features most responsible for differentiating methods of anesthesia and euthanasia in each tissue as determined by PLS-DA analysis.
Fig 4.
Heatmap illustrating alterations in metabolite levels in tissues collected using different methods of anesthesia.
Data are expressed as fold change versus cervical dislocation. n = 8 mice per method of anesthesia or euthanasia. • indicates p < 0.05 versus cervical dislocation after false discovery rate correction.
Fig 5.
Impact of anesthesia or euthanasia on selected metabolites.
Data are presented as mean ± standard error of the mean and expressed as peak area relative to cervical dislocation (CD) for each method of anesthesia or euthanasia. n = 8 mice per method of euthanasia. * indicates p < 0.05 versus cervical dislocation after false discovery rate correction.