n-3 Polyunsaturated Fatty Acids Reduce Neonatal Hypoxic/Ischemic Brain Injury by Promoting Phosphatidylserine Formation and Akt Signaling by Wenting Zhang, Jia Liu, Xiaoming Hu, Peiying Li, Rehana K. Leak, Yanqin Gao, and Jun Chen Stroke Volume 46(10):2943-2950 September 28, 2015 Copyright © American Heart Association, Inc. All rights reserved.

n-3 Polyunsaturated fatty acids protect against hypoxia/ischemia (H/I)–induced tissue loss and improve neurological outcomes. n-3 Polyunsaturated fatty acids protect against hypoxia/ischemia (H/I)–induced tissue loss and improve neurological outcomes. A, Left, Representative photographs of Cresyl Violet-stained coronal brain sections from N3L and N3H groups at 7 days post H/I. Right, Quantification of total tissue loss at 7 days post H/I (n=12 per group). *P≤0.05, **P≤0.01 vs N3L H/I. B, Micrographs showing histological changes in cortex (CTX), striatum (STR), and CA3 of the hippocampus (HIP) from lesioned (ipsilateral) and unlesioned (contralateral) sides. Scale bar, 25 μm. C, Performance during daily tests of gait (left) and the righting reflex (right) at 2 to 7 days after H/I (n=12 per group). D, Morris water maze results at 8 weeks after H/I (n=8 for each group): (a) latency to reach the platform in the learning phase of the test; (b) frequency of crossing the target quadrant in the memory phase; (c) swim speed during the probe trial; and (d) route traversed across the region of the platform (n=8). #P≤0.05 vs sham, *P≤0.05 vs N3L H/I at the same time point. Wenting Zhang et al. Stroke. 2015;46:2943-2950 Copyright © American Heart Association, Inc. All rights reserved.

n-3 Polyunsaturated fatty acids attenuate hypoxia/ischemia (H/I)–induced cell death. n-3 Polyunsaturated fatty acids attenuate hypoxia/ischemia (H/I)–induced cell death. A, Representative Western blot images of cleaved caspase-3 and cleaved caspase-9. B, Quantification of data is shown in (A). Cortical extracts were obtained from animals on N3L and N3H diets after sham operation or H/I (n=5 per condition). #P≤0.05 vs sham, *P≤0.05 vs N3L H/I at the same time point. C and D, Tissue sections from cerebral cortex were stained for cleaved caspase-3 (C) NeuN, TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) (D, top), or Fluoro-Jade B (D, bottom) at 24 hours after H/I (n=4 per group). Scale bar, 25 μm. Wenting Zhang et al. Stroke. 2015;46:2943-2950 Copyright © American Heart Association, Inc. All rights reserved.

Activation of Akt in neurons is critical for the neuroprotective effects of n-3 polyunsaturated fatty acids against hypoxia/ischemia (H/I). Activation of Akt in neurons is critical for the neuroprotective effects of n-3 polyunsaturated fatty acids against hypoxia/ischemia (H/I). A, Western blot analyses of phosphorylated Akt (p-Akt), Akt, p-GSK-3β, and GSK-3β in cortical extracts. Cortical extracts were obtained from animals on N3L and N3H diets after sham operation or H/I (n=6 per condition). #P≤0.05 vs sham, *P≤0.05 vs N3L H/I at the same time point. B, Representative coronal tissue sections immunostained for NeuN and p-Akt or labeled with Hoechst 33258 (n=4 per group). Scale bar, 20 μm. C and D, Five millilitre LY294002 (50 mmol/L) were injected during the course of 15 minutes at 1 hour before H/I. C, top, Western blots of p-Akt, total Akt, p-GSK-3β, total GSK-3β, and β-actin in cortical extracts at 24 hours after sham operation or H/I. Bottom, Quantification of p-Akt (left) and p-GSK-3β (right; n=4 per condition). D, Tissue loss at 7 days after H/I in N3L and N3H groups with or without LY294002 injection (n=14 per group). #P<0.05 vs sham, *P<0.05, **P<0.01. GSK indicates glycogen synthase kinase. Wenting Zhang et al. Stroke. 2015;46:2943-2950 Copyright © American Heart Association, Inc. All rights reserved.

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) directly protect primary cortical neurons. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) directly protect primary cortical neurons. A and B, Different concentrations of DHA (top) or EPA (bottom) were added to cultures 24 hours before 60 minutes oxygen-glucose deprivation (OGD). A, Lactate dehydrogenase (LDH) release was measured 24 hours after OGD. B, Cell viability was measured by quantification of live (green)/dead (red) cells 24 hours after OGD. Data are mean±SE from 3 experiments, each performed in triplicate. *P≤0.05; **P≤0.01 vs OGD alone. C, DHA (20 µmol/L) or EPA (10 μmol/L) was added to cultures 24 hours before 60 minutes OGD. Western blots of phosphorylated Akt (p-Akt) or total Akt at 2 or 6 hours after OGD (n=3 per group). D, The addition of a specific Akt inhibitor (Akt inhibitor IV, 3 µmol/L) 1 hour before OGD attenuated the neuroprotective effects of DHA and EPA against OGD. Cell viability was measured (quantification of live/dead staining) 24 hours after OGD (n=3 per group). #P≤0.05 vs vehicle, *P≤0.05 vs non-Akt inhibitor groups. Wenting Zhang et al. Stroke. 2015;46:2943-2950 Copyright © American Heart Association, Inc. All rights reserved.

Posphatidylserine (PS) is essential for docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)–mediated neuroprotection. Posphatidylserine (PS) is essential for docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)–mediated neuroprotection. A, PS content was measured in primary neurons at 24 hours after DHA or EPA treatment. *P≤0.05, **P≤0.01 vs control. B, Western blots of neuronal phosphorylated Akt (p-Akt) or total Akt at 2 and 6 hours after oxygen-glucose deprivation (OGD), after phosphatidylserine synthase-1 (PSS1) shRNA and scrambled shRNA transfection (n=4 per group). C, Cell viability was measured (quantification of live/dead staining) at 24 hours after OGD, after PSS1 shRNA and scrambled shRNA transfection (n=3 per group). #P≤0.05 vs OGD, *P≤0.05, **P≤0.01 vs PSS1sc group. D, Schematic illustration of the underlying mechanism of n-3 polyunsaturated fatty acids’ (PUFAs) neuroprotective effect in neurons. n-3 PUFAs enhance the biosynthesis of PS in neurons. PS on the neuronal membrane facilitates the membrane translocation of Akt in a PIP3-dependent manner, and enhances the subsequent phosphorylation and activation of Akt. PS may also directly interact with Akt and enhance its phosphorylation/activation. Activated Akt then inactivates the proapoptotic protein glycogen synthase kinase (GSK)-3β and suppresses neuronal cell death after ischemic damage. In addition, PS may participate in the activation of other prosurvival signaling molecules, such as PKC and Raf-1 kinase. n-3 PUFAs may also activate ERK 1/2 to enhance cell survival after hypoxia/ischemia. Wenting Zhang et al. Stroke. 2015;46:2943-2950 Copyright © American Heart Association, Inc. All rights reserved.