Article Figures & Data
Figures
- Figure 1.
Sirt1 activity increases after KA-SE in adult rats. ChIP was used to assess the binding of Sirt1 to chromatin, an indicator of its deacetylase activity. Hippocampi from KA-SE and control rats were obtained 1 h after KA-SE termination. We assessed specifically Sirt1 binding to the miR-124-1 gene promoter. A, Schematic of Sirt1 binding to miR-124-1 gene promoter and the locations of primer sequences. B, Sirt1 binding to the promoter of the miR-124-1 gene in KA-SE rats is significantly higher compared with that in CTRL rats (Student’s t test, *p < 0.05). Binding is shown as the percentage of input. The specificity of this binding is demonstrated by comparing it with the nonspecific binding of IgG to chromatin. TSS, Transcription start site; blue arrow, primer binding site (n = 4-6/group).
- Figure 2.
The Sirt1 inhibitor EX-527 blocks Sirt1 activity after KA-SE. A, B, Hippocampal mature miR-124 levels were measured by qPCR at 4 h after KA-SE termination in rats infused with vehicle or the Sirt1 inhibitor (EX-527) and normalized to GAPDH levels (A) or 14-3-3 ζ levels (B). A, KA-SE reduced miR-124 levels and EX-527 restored miR-124 levels (two-way ANOVA; main effect of inhibitor, p < 0.01; post hoc, **p < 0.01; n = 4/group). B, KA-SE reduced miR-124 levels and EX-527 restored miR-124 levels when normalized to 14-3-3 ζ (two-way ANOVA; main effect of inhibitor, p < 0.05; post hoc , *p < 0.05; n = 4/group).
- Figure 3.
Sirt1 inhibition does not prevent the development of epilepsy. Continuous digital video–EEG for 2 months was used to examine for spontaneous seizures in KA-SE rats infused with either vehicle (VEH) or Sirt1 inhibitor (EX-527) (n = 14/group). Most rats (26 of 28) developed spontaneous seizures (epilepsy) independent of treatment. A, The latency to the onset of the first seizure was not different between VEH- and EX-527-infused rats. B, The total number of seizures was not different between VEH- and EX-527-infused rats. C, The average number of seizures per day was not different between VEH- and EX-527-infused rats. D, Cumulative seizure numbers did not distinguish between groups. E, Median seizure duration did not differ between VEH- and EX-527-infused rats. F, Median seizure severity (using the Racine scale) was similar between VEH and EX-527 rats. G, Average frequency of interictal spike series per day did not distinguish the EX-527-treated and VEH-treated groups. H, The percentage of time spent in spike series after KA-SE was not different with EX-527 treatment (for spike series analysis: n = 6/group).
- Figure 4.
Sirt1 inhibition did not abrogate acute inflammation induced by KA-SE. Inflammatory markers in rat hippocampus were measured using qPCR at 4 h after KA-SE termination. A, COX-2 levels were strikingly increased by KA-SE; these levels were not affected by Sirt1 inhibitor EX-527 (two-way ANOVA; KA-SE main effect: F(1,12) = 204, p < 0.001; post hoc, ***p < 0.001). B, CCL3 mRNA levels were increased in the KA-SE groups, but EX-527 had no effect (KA-SE main effect: F(1,12) = 28, p < 0.001; post hoc, **p < 0.01,*p < 0.05). C, IL-1β levels were significantly reduced by the administration of EX-527 to control rats. The inhibitor did not influence cytokine IL-1β levels in KA-SE rats (two-way ANOVA; interaction of KA-SE and inhibitor: F(1,12) = 7, p < 0.05; post hoc, *p < 0.05). D, No significant changes in TNF-α were observed after EX-527 infusion (two-way ANOVA; trend for interaction of KA-SE and drug: F(1,12) = 4.4, p = 0.06; n = 4/group).
- Figure 5.
Cell loss after KA-SE was modest and was not affected by Sirt1 inhibition. NeuN straining was use to visualize neuronal dropout in the hippocampus 2 months after kainic acid–induced SE. A, Schematic of location for neuronal counts in the hilus and CA3 regions of the hippocampus. B, Representative images of average neuronal counts in the hilus region from CTRL and KA-SE rats treated with VEH or EX-527. C, Quantification of neuronal dropout in the hilus shows a small, insignificant loss of neurons in the hilus. D, Representative images of average neuronal counts in the CA3 region from CTRL and KA-SE rats treated with VEH or EX-527. E, Quantification of neurons in the CA3 region shows a small but insignificant loss of neurons in the CA3. (Controls, n = 4; KA-SE rats, n = 7/group).
Tables
mRNA Forward primer Reverse primer COX-2 TGGTGCCGGGTCTGATGATG GCAATGCGGTTCTGATACTG CCL3 GGGTGTCATTTTCCTGACCAAGAGAAACCG GCTGCCTCTAATCTCAGGCATTTAGTTCCAG IL-1β GTGAAATAGCAGCTTTCGACAGTGAGGAG GTGAGATTTGAAGCTGGATGCTCTCATCTG TNF-α CCCAGACCCTCACACTCAGAT TTGTCCCTTGAAGAGAACCTG GAPDH ATGCCATCACTGCCACTCAGA ACCAGTGGATGCAGGGATGAT 14-3-3 ζ ACCGTTACTTGGCCGAGGTT ACCGTTACTTGGCCGAGGTT Figure Data structure Statistical test Power/significance level 1B Normal distribution Unpaired t test 0.0317 2A Normal distribution Two-way ANOVA, Tukey’s test Drug, 0.0032
KA-SE, 0.8793
Interaction, 0.0642B Normal distribution Two-way ANOVA, Bonferroni’s test Drug, 0.02080
KA-SE, 0.0705
Interaction, 0.06253A Nonparametric Mann–Whitney test 0.2831 3B Nonparametric Mann–Whitney test 0.0990 3C Nonparametric Mann–Whitney test 0.3256 3D Nonparametric Mann–Whitney test 0.1429 3E Nonparametric Mann–Whitney test 0.7533 3G Nonparametric Mann–Whitney test 0.9004 3H Nonparametric Mann–Whitney test 0.4242 4A Normal distribution Two-way ANOVA, Tukey’s test Drug, 0.4538
KA-SE, <0.0001
Interaction, 0.54674B Normal distribution Two-way ANOVA, Tukey’s test Drug, 0.3588
KA-SE, 0.0002
Interaction, 0.10334C Normal distribution Two-way ANOVA, Tukey’s test Drug, 0.3638
KA-SE, 0.1229
Interaction, 0.05794D Normal distribution Two-way ANOVA, Tukey’s test Drug, 0.1461
KA-SE, 0.8726
Interaction, 0.0198
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