Brain oxylipin concentrations following hypercapnia/ischemia: effects of brain dissection and dissection time

Abstract

PUFAs are precursors to bioactive oxylipin metabolites that increase in the brain following CO₂-induced hypercapnia/ischemia. It is not known whether the brain-dissection process and its duration also alter these metabolites. We applied CO₂ with or without head-focused microwave fixation for 2 min to evaluate the effects of CO₂-induced asphyxiation, dissection, and dissection time on brain oxylipin concentrations. Compared with head-focused microwave fixation (control), CO₂ followed by microwave fixation prior to dissection increased oxylipins derived from lipoxygenase (LOX), 15-hydroxyprostaglandin dehydrogenase (PGDH), cytochrome P450 (CYP), and soluble epoxide hydrolase (sEH) enzymatic pathways. This effect was enhanced when the duration of postmortem ischemia was prolonged by 6.4 min prior to microwave fixation. Brains dissected from rats subjected to CO₂ without microwave fixation showed greater increases in LOX, PGDH, CYP and sEH metabolites compared with all other groups, as well as increased cyclooxygenase metabolites. In nonmicrowave-irradiated brains, sEH metabolites and one CYP metabolite correlated positively and negatively with dissection time, respectively. This study presents new evidence that the dissection process and its duration increase brain oxylipin concentrations, and that this is preventable by microwave fixation. When microwave fixation is not available, lipidomic studies should account for dissection time to reduce these artifacts.

Keywords: brain lipids; lipid mediators; polyunsaturated fatty acid metabolites.

Fig. 1.

Study design. Following 30 days of acclimatization, male Long Evans rats were randomly separated into 4 groups: group 1, MW + decapitation, in which rats were subjected to head-focused microwave fixation (13.5 kW for 1.6 s) prior to dissection (no ischemia; n = 8); group 2, CO₂ + MW + decapitation, in which rats were subjected to CO₂-induced asphyxiation for 2 min immediately followed by head-focused microwave fixation prior to dissection (n = 7); group 3, CO₂ + decapitation + MW, in which rats were subjected to CO₂-induced asphyxiation for 2 min, decapitated (and the decapitated heads left on the bench for an average of 6.38 min), and then subjected to microwave fixation prior to dissection (n = 8); and group 4, CO₂ + decapitation, in which rats were subjected to CO₂-induced asphyxiation for 2 min and then dissection (n = 8). The dissection time of group 3 (6.4 min) was matched to the dissection time of group 4 (6.6 min), which did not receive head-focused microwave irradiation. All brains were flash-frozen in liquid nitrogen after dissection. MW, microwave.

Fig. 2.

Partial least-squares discriminant analyses comparing oxylipin concentrations between the different experimental groups. The analysis resulted in a three-component model (Q² = 0.76; R² = 0.93). The MW + decapitation control group is represented in red (group 1), the CO₂ + MW + decapitation group is represented in dark blue (group 2), the CO₂ + decapitation + MW group is represented in light blue (group 3), and the CO₂ + decapitation group is represented in green (group 4). MW, microwave.

Fig. 3.

Heat map representing metabolites that differed significantly among the groups based on one-way ANOVA. The color scale from −3 to 3 represents the z-score, which was calculated by dividing the difference between the metabolite concentration value and the metabolite group mean by the overall SD. A positive z-score reflects increased metabolite concentrations (i.e., increased red color intensity); a negative z-score reflects decreased metabolite concentrations (i.e., decreased blue color intensity). The colors on top of the heat map are the color codes for each group (group 1, red; group 2, dark blue; group 3, light blue; and group 4, green). Their position shows how the groups cluster together, as determined by hierarchical clustering using Ward’s algorithm. Group 4 had the highest oxylipin concentrations, whereas group 1 had the lowest concentrations. Group 2 and group 3 clustered together with intermediate oxylipin concentrations. On the right side of the heat map are the metabolites themselves and their precursor fatty acid (e.g., ARA-derived 11-HETE). Metabolites in purple are derived from the COX pathway, those in orange are derived from the LOX pathway, those in pink are derived from the CYP pathway, and those in black are derived from the sEH pathway. The clustering of the PUFA-derived metabolites is depicted on the left side of the heat map. MW, microwave.

Fig. 4.

Significant Spearman’s correlation between oxylipin concentrations and dissection time in the CO₂ + decapitation group (group 4; n = 7). As outlined in Methods, the sample size was seven instead of eight because the duration of dissection was not collected for one rat.

Fig. 5.

Brains (500 mg) obtained from nonmicrowave-irradiated rats were homogenized in 400 µl extraction solvent containing antioxidants and surrogate standard. The brain samples used were remaining parts following the removal of the cortex, cerebellum, hippocampus, and brainstem for oxylipin analysis in a separate study (29). Homogenized samples were split into two: one sample was extracted directly, while the other was kept overnight at −80°C (with the brain homogenate) prior to extraction. Oxylipins derived from (A) LNA, (B) ARA, and (C) EPA, DHA, and DGLA were detected and quantified. Log-transformed data were analyzed by a paired t-test. Data are means ± SDs. There were no significant differences between the means (P > 0.05).