Brain 2-Arachidonoylglycerol Levels Are Dramatically and Rapidly Increased Under Acute Ischemia-Injury Which Is Prevented by Microwave Irradiation

Abstract

The involvement of brain 2-arachidonoylglycerol (2-AG) in a number of critical physiological and pathophysiological regulatory mechanisms highlights the importance for an accurate brain 2-AG determination. In the present study, we validated head-focused microwave irradiation (MW) as a method to prevent postmortem brain 2-AG alterations before analysis. We compared MW to freezing to prevent 2-AG induction and estimated exogenous and endogenous 2-AG stability upon exposure to MW. Using MW, we measured, for the first time, true 2-AG brain levels under basal conditions, 30 s after brain removal from the cranium, and upon exposure to 5 min of brain global ischemia. Our data indicate that brain 2-AG levels are instantaneously and dramatically increased approximately 60-fold upon brain removal from the cranium. With 5 min of brain global ischemia 2-AG levels are also, but less dramatically, increased 3.5-fold. Our data indicate that brain tissue fixation with MW is a required technique to measure both true basal 2-AG levels and 2-AG alterations under different experimental conditions including global ischemia, and 2-AG is stable upon exposure to MW.

Keywords: 2-Arachidonoylglycerol; Analysis; Brain; Endocannabinoids; Injury; Ischemia; Mass spectrometry; Microwave; Stroke.

Fig. 1

Extracted ion chromatograms for 2-AG and 2-AGd₈ analyzed using UPLC-MS/MS 2-AG were extracted from 10 mg of brain tissue with 100 ng of 2-AGd₈ added as an internal standard before extraction as described in the Methods. 0.5% of the extract was loaded on the column for UPLC-MS/MS analysis.

Fig. 2

A dramatic postmortem 2-AG increase is prevented by MW but not by fast freezing of intact head in liquid nitrogen Brain samples were analyzed from fixed with microwave irradiation brains (MW), from non-MW brains frozen within 30 sec after removing from the heads (non-MW), or from non-MW brains frozen in situ in the animal heads in liquid nitrogen within 3 sec after decapitation (non-MW, fast frozen). Samples were analyzed using UPLC-MS/MS analysis as described in the Methods. Values (also indicated above the bars) are mean ± SD, n=4. * - Statistically different (p<0.05) as compared to MW using ANOVA with Tukey post-hoc test. The data is representative of two independent sets of experiments.

Fig. 3

2-AG is rapidly produced and hydrolyzed upon incubation of non-MW brain tissue Frozen non-microwaved brains were pulverized and 10 mg of brain powder was spiked with 2-AGd₈ (10 µg). 2-AG and 2-AGd₈ were extracted immediately or after incubation at 40°C for 30 min and quantified using UPLC-MS/MS analysis as described in the Methods. Values (also indicated above the bars) are Mean ± SD, n=4. Because labeled 2-AGd₈ was used to account for hydrolysis of exogenous 2-AG, we could not use it as an internal standard. Thus, the results are reported as peak area for 2-AGd₈ and unlabeled 2-AG.* - Statistically different (p<0.05).

Fig. 4

MW is required to measure 2-AG alterations under ischemia/injury Rat brain 2-AG levels were analyzed immediately after decapitation (control) or after incubation of decapitated heads at 40°C for 5 min (ischemic). Before decapitation, brain tissue was fixed with head focused microwave irradiation (Microwaved) or processed without fixation (non-Microwaved). 2-AG was analyzed using UPLC-MS/MS analysis as described in the Methods. Values are mean ± SD, n=3. * - Statistically different (p<0.05).