Research ArticleNEUROSCIENCE

Elovanoids are a novel class of homeostatic lipid mediators that protect neural cell integrity upon injury

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Science Advances  27 Sep 2017:
Vol. 3, no. 9, e1700735
DOI: 10.1126/sciadv.1700735
  • Fig. 1 Discovery and structural characterization of ELVs (ELV-N32 and ELV-N34) in neuronal cell cultures.

    (A) The ELV structural framework was synthesized from three key intermediates a, b, and c, each of which was prepared from readily available starting materials. The stereochemistry of intermediates b and c was predefined by using enantiomerically pure epoxide starting materials. The final ELVs (d) were assembled via iterative couplings of intermediates a, b, and c, and they were isolated as the Me or Na. (B to K) Identification of ELVs in neuronal cell cultures. Cerebral-cortical mixed neuronal cells were incubated with 32:6n3 and 34:6n3 (10 μM each) under OGD conditions. In (B), 32:6n3 (red line), endogenous monohydroxy-32:6 (green line) and ELV-N32 (blue line) are shown with ELV-N32 standard (purple line) in the inset. MRM of ELV-N32 shows two large peaks eluted earlier than the peak when standard ELV-N32 is eluted, but they show the same fragmentation patterns, suggesting that they are isomers. In (C), the same features were shown in 34:6n3 and ELV-N34. (D) UV spectrum of endogenous ELV-N32 shows triene features, but these are not definite at this concentration. (E) Fragmentation pattern of ELV-N32. (F) UV spectrum of endogenous ELV-N34 showing triene features. (G) Fragmentation pattern of ELV-N34. (H) Full fragmentation spectra of endogenous ELV-N32 and (I) ELV-N32 standard show that all major peaks from the standard match to the endogenous peaks but are not perfectly matched; endogenous ELV-N32 has more fragments that do not show up in the standard, suggesting that it may contain isomers. (J) For ELV-N34 full fragmentation spectra, the endogenous ELV-N34 peaks match to the standard ELV-N34 (K), also suggesting the existence of ELV-N34 isomers.

  • Fig. 2 ELV-N32 and ELV-N34 elicit protection of cerebral-cortical mixed neuronal cell or hippocampal mixed neuronal cultures exposed to OGD or UOS.

    (A) Representative images of cerebral-cortical mixed neuronal cultures (DIV 12) challenged with 90-min OGD. The cells were fixed and stained with Hoechst 33258 after 12-hour treatment with ELV-N32 Na or ELV-N34 Me at a concentration of 500 nM, showing pyknosis as a result of OGD and neuroprotection elicited by ELV-N32 Na or ELV-N34 Me. (B) Summary of data from (A) [**P < 0.001 and ***P < 0.0001, one-way analysis of variance (ANOVA), followed by Holm-Sidak’s multiple comparisons test; n = 9]. (C and D) An unbiased image analysis method was applied to count Hoechst-positive nuclei, and the percentage of relative frequency distribution of pyknotic versus nonpyknotic nuclei is shown in the presence of OGD + ELV-N32 Na (C) or OGD + ELV-N34 Me (D), respectively. When the cells were subjected to OGD stress, they underwent pyknosis, as shown by the leftward shift of the nuclear peak. Again, upon treatment with either ELV-N32 Na or ELV-N34 Me, there was a positive rightward shift toward the control nuclear population peak, indicating that cellular survival was elicited by these novel lipid mediators. The nuclear size cutoff for defining pyknotic versus nonpyknotic nuclei is represented by black dashed lines and highlighted by a yellow rectangle. (E) Neuroprotection elicited by ELV-N32 Na or ELV-N34 Me, as assessed by calcein-positive cell counting after exposure of cerebral-cortical mixed neuronal cultures (DIV 12) challenged with 90-min OGD (****P < 0.0001, one-way ANOVA, followed by Holm-Sidak’s multiple comparisons test; n = 3). PI, propidium iodide. (F) Cell survival as assessed by MTT assay after exposure of cerebral-cortical mixed neuronal cell cultures (DIV 12) challenged with 90-min OGD, followed by treatment with ELV-N32 Me, ELV-N32 Na, ELV-N34 Me, or ELV-N34 Na at a concentration of 1 μM. (#P < 0.0001, *P < 0.05, and **P < 0.001, one-way ANOVA, followed by Holm-Sidak’s multiple comparisons test; n = 9). (G and H) Neuroprotection elicited by ELV-N32 Na, ELV-N34 Na, 32:6, or 34:6, as assessed by an unbiased image analysis followed by Hoechst-positive nuclei counting after hippocampal mixed neuronal culture (DIV 12), is subjected to OGD stress in the presence or absence of ELV-N32 Na or ELV-N34 Me at a concentration of 500 nM; 32:6 or 34:6 when added at a concentration of 500 nM also showed neuroprotection (#P < 0.0001, one-way ANOVA, followed by Holm-Sidak’s multiple comparisons test; n = 3) (G) or cerebral-cortical mixed neuronal cell culture (DIV 28) (#P < 0.0001, *P < 0.05, and **P < 0.001, one-way ANOVA, followed by Holm-Sidak’s multiple comparisons test; n = 3) (H), respectively, were subjected to 90-min OGD. (I) Neuroprotection elicited by ELV-N34 Na or ELV-N34 Me at a concentration of 200 nM, as assessed by an unbiased image analysis, followed by Hoechst-positive nuclei counting after cerebral-cortical mixed neuronal cell culture (DIV 12), were subjected to 12-hour UOS induced by the addition of TNFα (10 ng/ml) and H2O2 (50, 100, or 200 μM) (#P < 0.0001 and *P < 0.001, one-way ANOVA, followed by Holm-Sidak’s multiple comparisons test; n = 9).

  • Fig. 3 ELV-N32 and ELV-N34 induce protection of cerebral-cortical mixed neuronal cell cultures exposed to NMDA excitotoxicity.

    (A) Representative images of cerebral-cortical mixed neuronal cell cultures (DIV 12) subjected to 12-hour NMDA excitotoxicity. The cells were fixed and stained with Hoechst 33258 after 12-hour treatment with ELV-N32 Na or ELV-N34 Me at a concentration of 500 nM along with NMDA at a concentration of 100 μM, showing pyknosis as a result of NMDA excitotoxicity and neuroprotection elicited by ELV-N32 Na or ELV-N34 Me. (B) Summary of data from (A) (****P < 0.0001 and **P < 0.05, one-way ANOVA, followed by Holm-Sidak’s multiple comparisons test; n = 9). (C and D) An unbiased image analysis method was applied to count Hoechst-positive nuclei, and the percentage of relative frequency distribution of pyknotic versus nonpyknotic nuclei is shown in the presence of NMDA + ELV-N32 Na (C) or NMDA + ELV-N34 Me (D), respectively. When the cells were subjected to NMDA excitotoxicity, they underwent pyknosis, as shown by the leftward shift of the nuclear peak. Again, upon treatment with either ELV-N32 Na or ELV-N34 Me, there was a positive rightward shift toward the control nuclear population peak, indicating cellular survival elicited by these ELVs. The nuclear size cutoff for defining pyknotic versus nonpyknotic nuclei is represented by black dashed lines and highlighted by a yellow rectangle. (E) Neuroprotection elicited by ELV-N32 Na or ELV-N34 Me, as assessed by calcein-positive cell counting after exposure of cerebral-cortical mixed neuronal cell cultures (DIV 12) challenged with 12-hour NMDA at a concentration of 100 μM (****P < 0.0001, one-way ANOVA, followed by Holm-Sidak’s multiple comparisons test; n = 3). (F) Cell survival as assessed by MTT assay after exposing cerebral-cortical mixed neurons in culture (DIV 12) to NMDA (100 μM) excitotoxicity in the presence of the noncompetitive NMDA receptor antagonist MK801 maleate (dizocilpine) (10 μM), ELV-N32 Na (200 nM), or ELV-N32 Me (200 nM) (#P < 0.001, one-way ANOVA, followed by Holm-Sidak’s multiple comparisons test; n = 6). (G) Neuroprotective effects of ELV-N32 Na or ELV-N32 Me at a concentration of 200 nM after exposure of cerebral-cortical mixed neurons (DIV 12) to NMDA excitotoxicity (25, 50, or 100 μM). Cell survival was assessed by unbiased image analysis and counting of Hoechst-positive nuclei. (#P < 0.0001 and *P < 0.05, one-way ANOVA, followed by Holm-Sidak’s multiple comparisons test; n = 9). (H) Cerebral-cortical mixed neurons in culture (DIV 28) exposed to NMDA (50 μM) in the presence or absence of ELV-N32 Na or ELV-N34 Na or 32:6 or 34:6 (500 nM), as assessed by Hoechst staining and cell counting. (#P < 0.0001, one-way ANOVA, followed by Holm-Sidak’s multiple comparisons test; n = 3). ns, not significant.

  • Fig. 4 ELV-N32 and ELV-N34 improve neurological/behavioral score, protect the penumbra, and reduce MRI lesion volumes after ischemic stroke.

    (A) Total neurological score (normal score, 0; maximal score, 12) during MCAo (60 min) and at various times after treatment. At 60 min of MCAo, all animals had a score of 11 (of a possible 12). ELV-treated rats had significantly improved neurological scores on days 1, 3, and 7 compared to the vehicle (CSF)–treated group. (B) Ischemic core, penumbra, and total lesion volumes, computed from T2WI on day 7, were significantly reduced by ELV treatment compared to the vehicle group. (C) Representative T2WI, pseudo images, core/penumbra, and (D) 3D infarct volumes computed from T2WI on day 7. Core and penumbra were extracted from the entire brain. Core (red) and penumbral (blue) tissues were automatically identified in vehicle- and ELV-treated animals using the computational MRI method Hierarchical Region Splitting for penumbra identification. T2 hyperintensities were observed in the ischemic core and penumbra of vehicle-treated rats, consistent with edema formation. In contrast, ELV-treated animals had smaller lesion sizes. 3D reconstructions are from the same animal in each group on day 7. Values shown are means ± SD (n = 5 to 6 per group) (*P < 0.05, versus CSF group; repeated-measures ANOVA, followed by Bonferroni tests).

  • Fig. 5 ELV-N32 and ELV-N34 attenuate experimental ischemic stroke–induced neuronal and astrocyte cellular damage.

    (A and B) Representative NeuN (blue), SMI-71 (brown), and GFAP (dark green) stained brain sections from all groups. Vehicle (CSF)–treated rats showed extensive neuronal loss, reduction of GFAP-reactive astrocytes, and SMI-71–positive vessels. In contrast, treatment with ELVs increased NeuN-, SMI-71-, and GFAP-positive cells. (C) Coronal brain diagram (bregma, +1.2 mm) showing locations of regions for NeuN-, SMI-71-, and GFAP-positive cell counts in the cortex (a, b, and c) and striatum (s). Numbers of NeuN-positive neurons, SMI-71–positive vessels, and GFAP-positive astrocytes, increased by ELV treatment in the ischemic core (s) and different penumbral areas (a, b, and c), are shown. Values shown are means ± SD (*P < 0.05, significantly different from vehicle; repeated-measures ANOVA, followed by Bonferroni tests; n = 5 to 6 per group).

  • Fig. 6 ELV-N32 and ELV-N34 diminish NVU disruption and reduce brain infarction after ischemic stroke.

    (A) NVU breakdown was assessed by immunodetection of IgG within the parenchyma. IgG staining (brown) indicates NVU breakdown. Vehicle (CSF)–treated rats displayed increased IgG immunoreactivity in the cortex and subcortex. Treatment with ELV-N34 Na or ELV-N34 Me showed less IgG staining in the cortex and was mostly localized in the core of infarction (subcortex). (B) Bar graph shows that ELV-N34 Na and ELV-N34 Me significantly reduced IgG immunoreactivity in the cortex, subcortex, and whole hemisphere (total). Values shown are means ± SD (*P < 0.05, versus the vehicle group; repeated-measures ANOVA, followed by Bonferroni tests; n = 5 to 6 per group). (C) Nissl-stained brain sections from rats treated with vehicle and ELVs. Vehicle-treated rats show large cortical and subcortical infarction. In contrast, rats treated with ELVs show less extensive damage, mostly in the subcortical area. (D) Cortical, subcortical, and total corrected infarct volumes. All ELV treatments markedly reduced cortical, subcortical, and total infarct volumes compared to the vehicle-treated group. Values shown are means ± SD (*P < 0.05, significantly different from vehicle; repeated-measures ANOVA, followed by Bonferroni tests; n = 5 to 6 per group).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/9/e1700735/DC1

    fig. S1. ELV-N32 and ELV-N34 elicit protection of cerebral-cortical mixed neuronal cell cultures exposed to OGD or NMDA.

    fig. S2. ELV-N32 and ELV-N34 in neuronal cell cultures.

    fig. S3. Representative images of calcein-stained cerebral-cortical mixed neuronal cells exposed to OGD or NMDA.

    fig. S4. Representative bright-field and fluorescent images of cerebral-cortical mixed neuronal and hippocampal mixed neuronal cultures.

    fig. S5. Absolute frequency histograms of cerebral-cortical mixed neuronal or hippocampal mixed neuronal cultures exposed to OGD or NMDA.

    fig. S6. Percent relative frequency histograms.

    fig. S7. Clustered histograms of percent relative frequencies.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. ELV-N32 and ELV-N34 elicit protection of cerebral-cortical mixed neuronal cell cultures exposed to OGD or NMDA.
    • fig. S2. ELV-N32 and ELV-N34 in neuronal cell cultures.
    • fig. S3. Representative images of calcein-stained cerebral-cortical mixed neuronal cells exposed to OGD or NMDA.
    • fig. S4. Representative bright-field and fluorescent images of cerebral-cortical mixed neuronal and hippocampal mixed neuronal cultures.
    • fig. S5. Absolute frequency histograms of cerebral-cortical mixed neuronal or hippocampal mixed neuronal cultures exposed to OGD or NMDA.
    • fig. S6. Percent relative frequency histograms.
    • fig. S7. Clustered histograms of percent relative frequencies.

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