Rapid high-energy microwave fixation is required to determine the anandamide (N-arachidonoylethanolamine) concentration of rat brain.

Anandamide (N-arachidonoylethanolamine, AEA) is the putative endogenous ligand for the CB1 receptor. Despite being regulated enzymatically, brain AEA concentrations are quite variable and have been reported to increase in response to ischemia and post-mortem delay. Because these observations are similar to the effects of decapitation on brain concentrations of unesterified arachidonic acid and several of its metabolites, we propose that brain AEA concentrations also increase with decapitation and that immediate head-focused microwave irradiation is necessary to quantify basal brain AEA levels correctly. To test this hypothesis, we measured brain AEA levels in rats that were subjected to head-focused microwave irradiation 5 min. following decapitation (5.5 kW, 3.4 s) (ischemic) and prior to decapitation (controls). Brain AEA concentrations were quantified by LC/MS/MS. AEA concentrations from ischemic animals (10.01 +/- 4.41 pmol/g, mean +/- SD) were significantly higher and more variable than control concentrations (2.45 +/- 0.39 pmol/g). Thus, the basal concentration of AEA in the brain is lower than previously thought and future studies attempting to quantify brain AEA should consider using head-focused microwave fixation to prevent anomalous results.