XB-ART-55555
Mol Neurobiol
2018 Oct 01;5510:7822-7838. doi: 10.1007/s12035-018-0946-7.
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Neurobiological Correlates of Alpha-Tocopherol Antiepileptogenic Effects and MicroRNA Expression Modulation in a Rat Model of Kainate-Induced Seizures.
Ambrogini P
,
Albertini MC
,
Betti M
,
Galati C
,
Lattanzi D
,
Savelli D
,
Di Palma M
,
Saccomanno S
,
Bartolini D
,
Torquato P
,
Ruffolo G
,
Olivieri F
,
Galli F
,
Palma E
,
Minelli A
,
Cuppini R
.
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Seizure-triggered maladaptive neural plasticity and neuroinflammation occur during the latent period as a key underlying event in epilepsy chronicization. Previously, we showed that α-tocopherol (α-T) reduces hippocampal neuroglial activation and neurodegeneration in the rat model of kainic acid (KA)-induced status epilepticus (SE). These findings allowed us to postulate an antiepileptogenic potential for α-T in hippocampal excitotoxicity, in line with clinical evidence showing that α-T improves seizure control in drug-resistant patients. To explore neurobiological correlates of the α-T antiepileptogenic role, rats were injected with such vitamin during the latent period starting right after KA-induced SE, and the effects on circuitry excitability, neuroinflammation, neuronal death, and microRNA (miRNA) expression were investigated in the hippocampus. Results show that in α-T-treated epileptic rats, (1) the number of population spikes elicited by pyramidal neurons, as well as the latency to the onset of epileptiform-like network activity recover to control levels; (2) neuronal death is almost prevented; (3) down-regulation of claudin, a blood-brain barrier protein, is fully reversed; (4) neuroinflammation processes are quenched (as indicated by the decrease of TNF-α, IL-1β, GFAP, IBA-1, and increase of IL-6); (5) miR-146a, miR-124, and miR-126 expression is coherently modulated in hippocampus and serum by α-T. These findings support the potential of a timely intervention with α-T in clinical management of SE to reduce epileptogenesis, thus preventing chronic epilepsy development. In addition, we suggest that the analysis of miRNA levels in serum could provide clinicians with a tool to evaluate disease evolution and the efficacy of α-T therapy in SE.
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Species referenced: Xenopus
Genes referenced: tnf
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Fig. 1. Effect of α-tocopherol treatment on slice excitability in control and kainate-induced rats. a Field excitatory postsynaptic potential (fEPSP) recorded in CA1 pyramidal layer in treated kainate-exposed (αK) and untreated kainate-exposed (VK) rats. The arrowhead indicates multiple population spike recorded in VK rat slice. b fEPSP population spike number recorded in the experimental groups: αK, treated kainate-exposed; VK, untreated kainate-exposed; αC, treated non-epileptic; VC, untreated non-epileptic, in response to Schafferâs collaterals stimulation (increasing intensities from 20 to 160 pA). Statistical analyses performed by two-way ANOVA repeated measure, Tukeyâs post hoc: *pâ<â0.05; **pâ<â0.01. c Bath application of 50 μM BMI together with 50 μM 4-AP leads to the appearance of spontaneous interictal events: on the top, the enlarged view of a single event is reported. d Latency of the first interictal event recorded in the experimental groups. Statistical analyses performed by one-way ANOVA, Tukeyâs post hoc: *pâ<â0.05. All data are expressed as meanâ±âSEM | |
Fig. 2. Effect of α-tocopherol treatment on protein carbonyls (PCO) formation used as marker of oxidative stress. PCO content (nmol/mg protein) in the hippocampal homogenates obtained from rats of different experimental groups: αK, treated kainate-exposed; VK, untreated kainate-exposed; αC, treated non-epileptic; VC, untreated non-epileptic. Histograms represent PCO content of three independent measurements in each experimental group (meansâ±âSEM). Statistical analyses performed by one-way ANOVA and Tukeyâs post hoc test: **pâ<â0.01 | |
Fig. 3. Effect of α-tocopherol treatment on neuroinflammatory markers in control and kainate-induced epileptic rats. Western blot analysis of the expression levels of neuroinflammatory markers in hippocampal homogenates obtained from rats of the experimental groups: αK, treated kainate-exposed; VK, untreated kainate-exposed; αC, treated non-epileptic; VC, untreated non-epileptic. Per each marker protein: representative immunoblots are displayed and anti-actin blots are shown as loading control. Note that immunoblots are shown in the same sequence as bars in the corresponding histograms. Histograms represent densitometric analyses of blots from three independent experiments (meansâ±âSEM). Statistical analyses performed by one-way ANOVA and Tukeyâs post hoc test: *pâ<â0.05; **pâ<â0.01 | |
Fig. 4. Effect of α-tocopherol treatment on bloodâbrain barrier in control and kainate-induced epileptic rats. Western blot analysis of the expression levels of claudin-5, used as marker of bloodâbrain barrier integrity, in hippocampal homogenates obtained from rats of the experimental groups: αK, treated kainate-exposed; VK, untreated kainate-exposed; αC, treated non-epileptic; VC, untreated non-epileptic. Per each experimental group: representative immunoblots are displayed and anti-actin blots are shown as loading control. Note that immunoblots are shown in the same sequence as bars in the corresponding histograms. Histograms represent densitometric analyses of blots from three independent experiments (meansâ±âSEM). Statistical analyses performed by one-way ANOVA and Tukeyâs post hoc test: **pâ<â0.01 | |
Fig. 5. Effect of α-tocopherol treatment on miR expression. Quantification of miR-126, miR-146a, and miR-124 expression in hippocampal homogenates (a), and quantification of serum miR-126, miR-146a, and miR-124 expression (b) obtained from rats of the different experimental groups: αK, treated kainate-exposed; VK, untreated kainate-exposed; αC, treated non-epileptic; VC, untreated non-epileptic. Statistical analyses performed by one-way ANOVA and Tukeyâs post hoc test: *pâ<â0.05; **pâ<â0.01. All data have been normalized on VC miR expression level and expressed as meanâ±âSEM | |
Fig. 6. Effect of α-tocopherol treatment on miR-124 expression. Representative confocal FISH images of fluorescent in situ hybridization of miR-124 in CA1 pyramidal cells (scale bar 25 μm) (a). Quantification of FISH intensity of miR-124 normalized to U6 snRNA in CA1 pyramidal cells (b). αK, treated kainate-exposed; VK, untreated kainate-exposed; αC, treated non-epileptic; VC, untreated non-epileptic. All data are expressed as meanâ±âSEM. Statistical analyses performed by one-way ANOVA and Tukeyâs post hoc test: **pâ<â0.01 | |
Fig. 7. Effect of α-tocopherol treatment on neuronal degeneration. Histogram reports the quantification of FluoroJade-positive cells in CA1 hippocampal field in the different experimental groups. αK, treated kainate-exposed; VK, untreated kainate-exposed. Plate A, ââ3.3 mm; plate B, ââ4.3 mm; and plate C, ââ5.8 mm from bregma. Statistical analyses performed by Studentâs t test: **pâ<â0.01 |
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