A simplified method for preventing postmortem alterations of brain prostanoids for true in situ level quantification.

Dramatic postmortem prostanoid (PG) enzymatic synthesis in the brain causes a significant artifact during PG analysis. Thus, enzyme deactivation is required for an accurate in situ endogenous PG quantification. To date, the only method for preventing postmortem brain PG increase with tissue structure preservation is fixation by head-focused microwave irradiation (MW), which is considered the gold standard method, allowing for rapid in situ heat-denaturation of enzymes. However, MW requires costly equipment that suffers in reproducibility, causing tissue loss and metabolite degradation if overheated. Our recent study indicates that PGs are not synthesized in the ischemic brain unless metabolically active tissue is exposed to atmospheric O2. Based on this finding, we proposed a simple and reproducible alternative method to prevent postmortem PG increase by slow enzyme denaturation before craniotomy. To test this approach, mice were decapitated directly into boiling saline. Brain temperature reached 100°C after ∼140 s during boiling, though 3 min boiling was required to completely prevent postmortem PG synthesis, but not free arachidonic acid release. To validate this fixation method, brain basal and lipopolysaccharide (LPS)-induced PG were analyzed in unfixed, MW, and boiled tissues. Basal and LPS-induced PG levels were not different between MW and boiled brains. However, unfixed tissue showed a significant postmortem increase in PG at basal conditions, with lesser differences upon LPS treatment compared to fixed tissue. These data indicate for the first time that boiling effectively prevents postmortem PG alterations, allowing for a reproducible, inexpensive, and conventionally accessible tissue fixation method for PG analysis.

Exogenous oxygen is required for prostanoid induction under brain ischemia as evidence for a novel regulatory mechanism.

Previously, we and others reported a rapid and dramatic increase in brain prostanoids (PG), including prostaglandins, prostacyclins, and thromboxanes, under ischemia that is traditionally explained through the activation of esterified arachidonic acid (20:4n6) release by phospholipases as a substrate for cyclooxygenases (COX). However, the availability of another required COX substrate, oxygen, has not been considered in this mechanism. To address this mechanism for PG upregulation through oxygen availability, we analyzed mouse brain PG, free 20:4n6, and oxygen levels at different time points after ischemic onset using head-focused microwave irradiation (MW) to inactivate enzymes in situ before craniotomy. The oxygen half-life in the ischemic brain was 5.32 ± 0.45 s and dropped to undetectable levels within 12 s of ischemia onset, while there were no significant free 20:4n6 or PG changes at 30 s of ischemia. Furthermore, there was no significant PG increase at 2 and 10 min after ischemia onset compared to basal levels, while free 20:4n6 was increased ∼50 and ∼100 fold, respectively. However, PG increased ∼30-fold when ischemia was followed by craniotomy of nonMW tissue that provided oxygen for active enzymes. Moreover, craniotomy performed under anoxic conditions without MW did not result in PG induction, while exposure of these brains to atmospheric oxygen significantly induced PG. Our results indicate, for the first time, that oxygen availability is another important regulatory factor for PG production under ischemia. Further studies are required to investigate the physiological role of COX/PG regulation through tissue oxygen concentration.

12-Hydroxyeicosatetraenoic acid is the only enzymatically produced HETE increased under brain ischemia.

Hydroxyeicosatetraenoic acids (HETE) are dramatically increased under brain ischemia and significantly affect post-ischemic recovery. However, the exact mechanism of HETE increase and their origin under ischemia are poorly understood. HETE might be produced de novo through lipoxygenase (LOX) -dependent synthesis with possible esterification into a lipid storage pool, or non-enzymatically through free radical oxidation of esterified arachidonic acid (20:4n6). Because HETE synthesized through LOX exhibit stereospecificity, chiral analysis allows separation of enzymatic from non-enzymatic pools. In the present study, we analyzed free HETE stereoisomers at 30 sec, 2 min, and 10 min of ischemia. Consistent with previous reports, we demonstrated a significant, gradual increase in all analyzed HETE over 10 min of brain ischemia, likely attributed to release of the esterified pool. The R/S ratio for 5-HETE, 8-HETE, and 15-HETE was not different from a racemic standard mix, indicating their non-enzymatic origin, which was in opposition to the inflamed tissue used as a positive control in our study. However, 12(S)-HETE was the predominant isoform under ischemia, indicating that ∼90 % of 12-HETE are produced enzymatically. These data demonstrate, for the first time, that 12-LOX is the major LOX isoform responsible for the enzymatic formation of the inducible HETE pool under ischemia. We also confirmed the requirement for enzyme inactivation with high-energy focused microwave irradiation (MW) for accurate HETE quantification and validated its application for chiral HETE analysis. Together, our data suggest that 12-LOX and HETE-releasing enzymes are promising targets for HETE level modulation upon brain ischemia.