Exacerbation of ischemic injury in CBP^+/− primary cortical cultures.

<p><b>A</b>, Cortical neurons isolated from CBP^+/− mice or wild-type littermates were exposed at DIV 9 (<i>in vitro</i> day 9) to oxygen-glucose deprivation (OGD) for 75, 95 or 115 min. Lactate dehydrogenase (LDH) release was measured after 24 h as a marker to quantify neuronal death. Bar graphs represent the average LDH release of 4 independent experiments ± SEM. **<i>p</i><0.01; *<i>p</i><0.05. <b>B</b>, Representative result of Western blotting analyses of CBP^+/− mice and wild-type littermate cortical cultures using antibodies against ac-histone H4, ac-histone H4 and actin. <b>C</b>, Quantification of the Western blotting images was performed using ImageJ program and t test was conducted for statistical analyses. Bar graphs represent the mean values from 2 experiments ± SEM. *<i>p</i><0.05.</p

Injurious ischemia in primary cortical cultures induces a strong reduction in histone acetylation and a rapid decrease in CBP protein levels.

<p><b>A</b>, Rat cortical neurons were exposed at DIV 9 (<i>in vitro</i> day 9) to oxygen-glucose deprivation (OGD) for 150 min; 24 h after the injurious treatment, neuronal death was monitored by determining the release of lactate dehydrogenase (LDH) in culture medium. Bar graphs represent the average LDH release of 4 independent experiments ± SEM. ***<i>p</i><0.0001. <b>B</b>, Representative result of Western blotting analyses of neuronal cultures at various time points following injurious OGD using antibodies against ac-histone H4, ac-histone H4 and actin. <b>C</b>, Quantification of Western blotting images was performed using ImageJ program and one-way ANOVA followed by Tukey test was conducted for statistical analyses. Bar graphs represent the mean values from 3 experiments ± SEM. *<i>p</i><0.05. <b>D</b>, Representative result of Western blotting analyses of rat cortical neurons following injurious oxygen-glucose deprivation (OGD) for 150 min at DIV 9 (<i>in vitro</i> day 9) using antibodies against CBP and actin. E, Quantification of the Western blotting images was performed using ImageJ program and one-way ANOVA followed by Tukey test was conducted for statistical analyses. Bar graphs represent the mean values from 3 experiments ± SEM. *<i>p</i><0.05. F, Representative images of immunocytochemical staining of cortical neurons that were fixed immediately after 150 min injurious OGD. Nuclear staining (green), CBP (red). Sytox Green dye was used for DNA counterstaining of nucleic acids. Scale bar, 30 µm. The images are representative results of 3 independent experiments.</p

Exacerbation of ischemic injury in CBP^+/− primary cortical cultures.

<p><b>A</b>, Cortical neurons isolated from CBP^+/− mice or wild-type littermates were exposed at DIV 9 (<i>in vitro</i> day 9) to oxygen-glucose deprivation (OGD) for 75, 95 or 115 min. Lactate dehydrogenase (LDH) release was measured after 24 h as a marker to quantify neuronal death. Bar graphs represent the average LDH release of 4 independent experiments ± SEM. **<i>p</i><0.01; *<i>p</i><0.05. <b>B</b>, Representative result of Western blotting analyses of CBP^+/− mice and wild-type littermate cortical cultures using antibodies against ac-histone H4, ac-histone H4 and actin. <b>C</b>, Quantification of the Western blotting images was performed using ImageJ program and t test was conducted for statistical analyses. Bar graphs represent the mean values from 2 experiments ± SEM. *<i>p</i><0.05.</p

Reduction of CBP in cortical neurons by miR-shRNA treatment exacerbates ischemic injury responses.

<p><b>A</b>, Mouse cortical neurons were transduced at DIV 3 (<i>in vitro</i> day 3) with lentivirus-expressing CBP-specific miR-shRNA. At DIV 9, cultures were exposed to oxygen-glucose deprivation (OGD). Representative photomicrographs showing GFP expression that were taken shortly before and 24 h after OGD from the same microscopic fields. Scale bar, 100 µm. <b>B</b>, Representative result of Western blotting analyses of cortical neurons at DIV 9 after transduction with lentivirus-expressing miR-shRNAs using antibodies against CBP and actin. Reduction in CBP protein levels is particularly observable in cultures transduced with miR-shRNA-2 and miR-shRNA-3. <b>C</b>, Quantification of the Western blotting images was performed using ImageJ program and one-way ANOVA followed by Tukey test was conducted for statistical analyses. Bar graphs represent the mean values from 3 experiments ± SEM. <b>D</b>, Cortical neurons were transduced with the indicated lentiviral particles and analyzed after 75 and 115 min OGD. Survival of GFP-positive neurons exploited cell counts of pre- and post-OGD photomicrographs with mean data pooled from 3 independent experiments ± SEM. ***<i>p</i><0.001; **<i>p</i><0.01. <b>E</b>, Cortical neurons were transduced with the indicated miR-shRNAs and subjected to 115 min of injurious oxygen-glucose deprivation (OGD). Neuronal cell death was monitored by the release of lactate dehydrogenase measurement (LDH) from 3 independent experiments ± SEM. ***<i>p</i><0.001.</p

Increased histone H4 acetylation and recruitment of CBP to gelsolin promoter region precedes transcriptional upregulation of gelsolin after ischemic preconditioning.

<p><b>A</b>, Rat cortical neurons were subjected to 30 min non-injurious preconditioning OGD and analyzed by chromatin immunoprecipitation (ChIP) with the indicated antibodies after 1 h interval following the preconditioning. Real time PCR results for gelsolin promoter were presented as mean fold enrichment ± SEM. **<i>p</i><0.01; *<i>p</i><0.05. N = 4 experiments. <b>B</b>, Rat cortical neurons were subjected to 30 min non-injurious preconditioning OGD or control treatment and analyzed by semi-quantitative real-time RT-PCR for gelsolin at the indicated time points. The data is shown as mean ± SEM. *p<0.05 versus vehicle. N = 3 experiments.</p

Changes in histone acetylation in cortical neurons after ischemic preconditioning.

<p><b>A</b>, Experimental design. Rat cortical neurons were exposed to 30 min non-injurious preconditioning oxygen-glucose deprivation (OGD) on DIV 9 (<i>in vitro</i> day 9) and, 24 h afterwards, subjected to 150 min injurious damaging OGD. Neuronal cell death was monitored by lactate dehydrogenase analysis (LDH) further 24 h later. <b>B</b>, Representative result of Western blotting analyses of neuronal cultures at various time points after preconditioning using antibodies against ac-histone H4, ac-histone H3 and actin. <b>C</b>, Quantification of the Western blotting images was performed using ImageJ program and one-way ANOVA followed by Tukey test was conducted for statistical analyses. Bar graphs represent the mean values from 4 experiments ± SEM. *<i>p</i><0.05. <b>D</b>, Neuronal death was assessed by LDH measurement as a marker for neuronal death in preconditioned and non-preconditioned cultures after their exposure to 150 min injurious OGD. Thirty min preconditioning OGD reduced LDH levels significantly, providing protection against 150 min injurious OGD. Bar graphs represent the average LDH release of 4 independent experiments ± SEM. *<i>p</i><0.001.</p

Curcumin, a CBP HAT activity inhibitor, reduces histone acetylation and attenuates ischemic preconditioning in primary cortical cultures.

<p><b>A</b>, Representative result of Western blotting analyses of neuronal cultures following treatment with indicated concentrations of Curcumin for 24 h using antibodies against ac-histone H4, ac-histone H4 and actin. <b>B</b>, Quantification of the Western blotting images was performed using ImageJ program and one-way ANOVA followed by Tukey test was conducted for statistical analyses. Bar graphs represent the mean values from 4 experiments ± SEM. *<i>p</i><0.05. <b>C</b>, Experimental design. Rat cortical neurons were exposed to 30 min non-injurious preconditioning oxygen-glucose deprivation (OGD) on DIV 9 (<i>in vitro</i> day 9), and treated with 1 µM curcumin for the 24 h interval between the preconditioning and injurious OGDs. Damaging OGD of 150 min was conducted on DIV 10, and neuronal cell death was monitored by lactate dehydrogenase analysis (LDH) further 24 h later. <b>D</b>, Neuronal death was assessed 24 h after the injurious OGD exploiting the increase in LDH in the culture medium. Curcumin per se had no significant impact on the release of LDH in controls or OGD treated cultures which were not exposed to preceding preconditioning OGD. *p<0.05, unpaired t-test. The graph shows LDH release in preconditioned cultures relative to non-preconditioned cultures for vehicle and curcumin treatment conditions. N = 2 experiments. Data is shown as scattered dot blots representing cell culture wells with mean relative protection ± SEM.</p