Fig 1.
24S-HC exacerbates OGD-induced, NMDAR-dependent cell death.
(A) Representative images showing propidium iodide (PI)–labeled, compromised cells in control, OGD, OGD+24S-HC (2 μM), and OGD+24S-HC+APV (50 μM). Scale bar is 30 μm. (B) Survival rate was calculated as number of dead cells normalized to total cells and compared between control, OGD, OGD+APV, OGD+24S-HC, and OGD+24S-HC+APV. Cells treated with OGD and 24S-HC exhibited significantly lower survival rate compared to those treated with OGD alone (n = 7 cultures for each group; one-way repeated measures ANOVA with Bonferroni’s post hoc test, *P < 0.05). (C) 24S-HC levels in the culture media were measured from control (no virus infection), AAV8-GFP, and AAV8-CYP46A1-GFP infected cell cultures (n = 4 cultures for each group; one-way repeated measures ANOVA and Bonferroni post hoc test, *P < 0.05). (D) Representative images showing PI-labeled dead cells in GFP-expressing cells and CYP46A1 GFP-expressing cells with or without OGD challenges. Scale bar is 30 μm. (E) Survival rate was compared between control GFP and CYP46A1 GFP-expressing cells with/without OGD, and with OGD+ APV. Following OGD treatment, CYP46A1 GFP-expressing cells had significantly lower survival rate compared to GFP-expressing cells (n = 5 cultures for each group; two-way repeated measures ANOVA with paired t test, *P <0.05).
Fig 2.
Endogenous 24S-HC exacerbates OGD-induced, NMDAR-dependent cell death.
(A) Survival rate was compared between WT and KO cells with/without OGD challenges, and with OGD/APV co-treatment. Following OGD treatment, KO cells had significantly higher survival rate compared to WT cells (n = 5 cultures for WT and 5 cultures for KO; one-way ANOVA with Bonferroni’s post hoc test, *P < 0.05). (B) 24S-HC concentration (ng/ml) was measured in MEM and the original conditioned medium (CM) immediately following OGD insult and one hour after, respectively. (C) 24S-HC levels measured both immediately following OGD insult and one hour after insult, as shown in (B), were normalized to 24S-HC measured in the sibling untreated control cultures shown in (B) (n = 5 for control cultures and 6 for OGD cultures; P > 0.2).
Fig 3.
The 24S-HC exacerbation of OGD damage is concentration dependent and is partially rescued by 25-HC.
(A) Survival was compared between control, OGD, OGD+50 nM 24S-HC, OGD+0.5 μM 24S-HC, OGD+2 μM 24S-HC, and OGD+2 μM 24S-HC+10 μM 25-HC. Cells treated with OGD and 24S-HC at 0.5 μM and 2 μM showed significantly poorer survival compared to those treated with OGD alone. Application of 25-HC partially prevented OGD-induced cell death exacerbated by 2 μM 24S-HC. (n = 11 cultures for each group; one-way repeated measures ANOVA with Bonferroni’s post hoc test, *P < 0.05; **P < 0.01; ***P<0.001). (B) 25-HC not only partially rescued OGD-induced cell death following SGE-201 application, but also protected against OGD-induced cell death in the absence of 24S-HC analogue application (n = 8 cultures for each group; one-way repeated measures ANOVA with Bonferroni’s post hoc test, *P < 0.05; **P < 0.01; ***P < 0.001).
Fig 4.
25-HC alone is neuroprotective against OGD-induced cell death, independent of NMDARs.
(A) Survival rates from cultures treated with APV before or after OGD insult were normalized to the survival rate of cultures treated with OGD alone. APV (50 μM) administered before OGD insult had significantly larger neuroprotective effects on the survival rate compared to APV administered after OGD insult (n = 7 cultures for APV-before; n = 15 cultures for APV-after; *P < 0.05). (B) Survival rates from cultures treated with 25-HC before or after OGD insult were normalized to the survival rate of cultures treated with OGD alone. Administration of 25-HC (10 μM) before and after OGD insult had indistinguishable rescuing effects on survival (n = 15 cultures for each group; P > 0.2).
Fig 5.
25-HC alone does not protect against NMDA-induced cell death, but it alleviates 24S-HC exacerbated toxicity.
(A) Application of NMDA at 8 μM and 20 μM significantly reduced cell survival, which was not inhibited when cells were co-treated with 10 μM 25-HC (n = 13 cultures for each group, one-way repeated measures ANOVA with Bonferroni’s post hoc test, ***P < 0.001). (B) Application of 24S-HC significantly exacerbated 8 μM NMDA-induced cell death. The exacerbation was inhibited by 10 μM 25-HC (n = 6 cultures for each group, one-way repeated measures ANOVA with Bonferroni’s post hoc test, *P < 0.05).
Fig 6.
25-HC has a species-dependent, NMDAR-independent protecting effect on OGD-induced cell death.
(A) Survival rate in rat cultures was compared between control, OGD (3 hr), OGD (3 hr) + MK-801(20 μM), and OGD (3 hr) + MK-801(20 μM)+ 25-HC (20 μM). Cell death induced by more severe OGD (3 hr) was only partially inhibited by MK-801 application. 25-HC protected against the MK-801 insensitive OGD-induced cell death. (n = 12 cultures for each group, one-way repeated measures ANOVA with Sidak’s post hoc test, **P < 0.01, ***P < 0.001). As in other experiments with 25-HC, neuroprotection was modest but reliable across experiments. (B) SGE-201 did not increase cell death, verifying the NMDAR independence (N = 9 cultures for each group; P > 0.05). (C) H2O2 (100 μM, 1 hour treatment) toxicity was used to test neuroprotection of 25-HC. 25-HC again yielded mild but reliable protection (N = 10 cultures for each group, one-way repeated measures ANOVA with Bonferroni’s post hoc test, *P < 0.05). (D) and (E) Neither WT nor CYP46A1 KO hippocampal neuron cultures from C57Bl/6 mice was sensitive to the mild NMDAR independent neuroprotective effect of 25-HC (N = 7 cultures for each group; P > 0.05).