Effects of Anesthetic Administration on Rat Hypothalamus and Cerebral Cortex Peptidome

Abstract

Prohormone-derived neuropeptides act as cell-cell signaling molecules to mediate a wide variety of biological processes in the animal brain. Mass spectrometry-based peptidomic experiments are valuable approaches to gain insight into the dynamics of individual peptides under different physiological conditions or experimental treatments. However, the use of anesthetics during animal procedures may confound experimental peptide measurements, especially in the brain, where anesthetics act. Here, we investigated the effects of the commonly used anesthetics isoflurane and sodium pentobarbital on the peptide profile in the rodent hypothalamus and cerebral cortex, as assessed by label-free quantitative peptidomics. Our results showed that neither anesthetic dramatically alters peptide levels, although extended isoflurane exposure did cause changes in a small number of prohormone-derived peptides in the cerebral cortex. Overall, our results demonstrate that acute anesthetic administration can be utilized in peptidomic experiments of the hypothalamus and cerebral cortex without greatly affecting the measured peptide profiles.

Keywords: Neuropeptides; anesthetics; cerebral cortex; hypothalamus; mass spectrometry; peptidomics.

Figure 1.

PCA scores plot for peptide abundance data after preprocessing and normalization by EigenMS in the (A) hypothalamus or (B) cerebral cortex. For each tissue, n = 46 animals were analyzed, split into “No anesthesia” (n = 12), “Sodium pentobarbital (<6 min)” (n = 11), “Isoflurane (<6 min)” (n = 12), or “Isoflurane (60 min)” (n = 11) groups.

Figure 2.

Volcano plots comparing hypothalamus peptide changes for (A-C) each anesthesia group relative to “No anesthesia” group, or (D-F) each anesthesia group relative to each other. Red and blue data points indicate significantly changing peptides (passing both parametric and non-parametric tests with p < 0.05, and where p_adj is the minimum p value from these tests). Red data points show higher abundance and blue data points show lower abundance in the first group in each comparison. The two vertical lines are demarcation points for the log₂(fold-change) > 0.6 or < −0.6 between the groups. The horizontal line marks a p-value of 0.05. Black points above this horizontal line did not pass both the parametric and non-parametric tests.

Figure 3.

Volcano plots comparing cerebral cortex peptide changes for (A-C) each anesthesia group relative to “No anesthesia” group, and (D-F) each anesthesia group relative to each other. Red and blue data points indicate significantly changing peptides (passing both parametric and non-parametric tests with p < 0.05, and where p_adj is the minimum p value from these tests). Red data points show higher abundance and blue data points show lower abundance in the first group in each comparison. The two vertical lines are demarcation points for the log₂(fold-change) > 0.6 or < −0.6 between the groups. The horizontal line marks a p-value of 0.05. Black points above this horizontal line did not pass both the parametric and non-parametric tests.