Linoleic acid participates in the response to ischemic brain injury through oxidized metabolites that regulate neurotransmission

Abstract

Linoleic acid (LA; 18:2 n-6), the most abundant polyunsaturated fatty acid in the US diet, is a precursor to oxidized metabolites that have unknown roles in the brain. Here, we show that oxidized LA-derived metabolites accumulate in several rat brain regions during CO₂-induced ischemia and that LA-derived 13-hydroxyoctadecadienoic acid, but not LA, increase somatic paired-pulse facilitation in rat hippocampus by 80%, suggesting bioactivity. This study provides new evidence that LA participates in the response to ischemia-induced brain injury through oxidized metabolites that regulate neurotransmission. Targeting this pathway may be therapeutically relevant for ischemia-related conditions such as stroke.

Figure 1

Heat map of oxylipin concentrations in cortex, hippocampus, cerebellum and brainstem in control and ischemic rats. EET, epoxyeicosatrienoic acid; PG, prostaglandin; TXB2, Tromboxane B2; HETE, hydroxyeicosatetraenoic acid; oxo-ETE, oxo-eicosatetraenoic acid; DiHETE, dihydroxyeicosatetraenoic acid; LTB4, leukotriene B4; EpDPE, epoxydocosapentaenoic acid; DiHDPE, dihydroxydocosapentaenoic acid; HDoHE, hydroxydocosahexaenoic acid; HODE, hydroxyoctadecadienoic acid; oxo-ODE, oxo-octadecadienoic acid; EpOME, epoxyoctadecamonoenoic acid; diHOME, dihydroxyoctadecamonoenoic acid; TriHOME, trihydroxyoctadecamonoenoic acid; EpODE, epoxyoctadecadienoic acid; HETrE, hydroxyeicosatrienoic acid.

Figure 2

Cortex (a), hippocampus (b), cerebellum (c) and brainstem (d) linoleic acid (LA)-derived metabolite concentrations (in pmol/g) in microwave (MW) control and ischemic rats (CO₂; n = 7–9 per group). Values are mean ± standard deviation (SD). Significant differences were assessed using an unpaired t-test (*p < 0.05; **p < 0.01; ***p < 0.001). HODE, hydroxyoctadecadienoic acid; oxo-ODE, oxo-octadecadienoic acid; EpOME, epoxyoctadecamonoenoic acid; DiHOME, dihydroxyoctadecamonoenoic acid; TriHOME, trihydroxyoctadecamonoenoic acid; THF, tetrahydrofuran; EKODE, epoxyketooctadecadienoic acid.

Figure 3

Cortex (a), hippocampus (b), cerebellum (c) and brainstem (d) arachidonic acid (AA)-derived metabolite concentrations (in pmol/g) in microwave (MW) control and ischemic rats (CO₂; n = 7–9 per group). Values are mean ± standard deviation (SD). Significant differences were assessed using an unpaired t-test (*p < 0.05; **p < 0.01; ***p < 0.001). EET, epoxyeicosatrienoic acid; DiHETrE, dihydroxyeicosatrienoic acid; TriHETrE, trihydroxyeicosatrienoic acid; PG, prostaglandin; TXB2, thromboxane B2; HETE, hydroxyeicosatetraenoic acid; oxo-ETE, oxo-eicosatetraenoic acid; DiHETE, dihydroxyeicosatetraenoic acid; LTB4, leukotriene B4; LXA4, lipoxins A4.

Figure 4

Cortex (a), hippocampus (b), cerebellum (c) and brainstem (d) docosahexaenoic acid (DHA)-derived metabolite concentrations (in pmol/g) in microwave (MW) control and ischemic rats (CO₂; n = 7–9 per group). Values are mean ± standard deviation (SD). Significant differences were assessed using an unpaired t-test (*p < 0.05; **p < 0.01; ***p < 0.001). EpDPE, epoxydocosapentaenoic acid; DiHDPE, dihydroxydocosapentaenoic acid; HDoHE, hydroxydocosahexaenoic acid.

Figure 5

Cortex (a), hippocampus (b), cerebellum (c) and brainstem (d) di-homo-gamma-linolenic acid (DGLA)-, α-linolenic acid (ALA)- and eicosapentaenoic acid (EPA)-derived metabolite concentrations (in pmol/g) in microwave (MW) control and ischemic rats (CO₂; n = 7–9 per group). Values are mean ± standard deviation (SD). Significant differences were assessed using an unpaired t-test (*p < 0.05; **p < 0.01; ***p < 0.001). PG, prostaglandin; HETrE, hydroxyeicosatrienoic acid; EpODE, epoxyoctadecadienoic acid; DiHODE, dihydroxyoctadecadienoic acid; HEPE, hydroxyeicosapentaenoic acid; DiHETE, dihydroxyeicosatetraenoic acid; EpETE, epoxyeicosatetraenoic acid; HOTrE, hydroxyoctadecatrienoic acid.

Figure 6

Somatic (a and b) and dendritic (c and d) Paired-Pulse Facilitation (PPF) measured on hippocampal slices perfused with vehicle (artificial cerebrospinal fluid containing 0.1% ethanol), 1 µM linoleic acid (LA), 1 µM arachidonic acid (AA), 0.1 µM or 1 µM 13-hydroxyoctadecadienoic acid (13-HODE), or 0.1 µM or 1 µM prostaglandin E₂ (PGE₂). Data (mean ± SD) are expressed relative to baseline (n = 4–6 per condition). Graphs (a and c) showe the minute-by-minute data; (b and d) represent average PPF during compound incubation and washout relative to baseline (dotted line). Data were analyzed by a two-way repeated measures ANOVA followed by Dunnett’s multiple comparison test. *Significantly different compared to vehicle at a specific time point.

Figure 7

Dynamic multiple reaction monitoring scan of oxidized fatty acids in artificial cerebrospinal fluid (ACSF) containing vehicle, 1 µM linoleic acid (LA), 1 µM arachidonic acid (AA), 1 µM 13-hydroxyoctadecadienoic acid (13-HODE), or 1 µM prostaglandin E₂ (PGE₂). The vehicle or compounds were incubated in ACSF at 37 °C for 10 min under constant bubbling of 95% O₂. No contamination was observed in vehicle and ACSF containing LA and AA. ACSF containing 13-HODE was pure at >98%. ACSF containing PGE₂ had 20% PGE₂, 78% PGD₂ and 2% unidentified impurities. As shown in the figure, PGE₂ and PGD₂ peaks eluted at the same time.