Pioglitazone blocks ethanol induction of microglial activation and immune responses in the hippocampus, cerebellum, and cerebral cortex in a mouse model of fetal alcohol spectrum disorders - PubMed (original) (raw)

Pioglitazone blocks ethanol induction of microglial activation and immune responses in the hippocampus, cerebellum, and cerebral cortex in a mouse model of fetal alcohol spectrum disorders

Paul D Drew et al. Alcohol Clin Exp Res. 2015 Mar.

Abstract

Background: Fetal alcohol spectrum disorders (FASD) result from fetal exposure to alcohol and are the leading cause of mental retardation in the United States. There is currently no effective treatment that targets the causes of these disorders. Thus, novel therapies are critically needed to limit the neurodevelopmental and neurodegenerative pathologies associated with FASD.

Methods: A neonatal mouse FASD model was used to examine the role of the neuroimmune system in ethanol (EtOH)-induced neuropathology. Neonatal C57BL/6 mice were treated with EtOH, with or without pioglitazone, on postnatal days 4 through 9, and tissue was harvested 1 day post treatment. Pioglitazone is a peroxisome proliferator-activated receptor (PPAR)-γ agonist that exhibits anti-inflammatory activity and is neuroprotective. We compared the effects of EtOH with or without pioglitazone on cytokine and chemokine expression and microglial morphology in the hippocampus, cerebellum, and cerebral cortex.

Results: In EtOH-treated animals compared with controls, cytokines interleukin-1β and tumor necrosis factor-α mRNA levels were increased significantly in the hippocampus, cerebellum, and cerebral cortex. Chemokine CCL2 mRNA was increased significantly in the hippocampus and cerebellum. Pioglitazone effectively blocked the EtOH-induced increase in the cytokines and chemokine in all tissues to the level expressed in handled-only and vehicle-treated control animals. EtOH also produced a change in microglial morphology in all brain regions that was indicative of microglial activation, and pioglitazone blocked this EtOH-induced morphological change.

Conclusions: These studies indicate that EtOH activates microglia to a pro-inflammatory stage and also increases the expression of neuroinflammatory cytokines and chemokines in diverse regions of the developing brain. Further, the anti-inflammatory and neuroprotective PPAR-γ agonist pioglitazone blocked these effects. It is proposed that microglial activation and inflammatory molecules expressed as a result of EtOH treatment during brain development contribute to the sequelae associated with FASD. Thus, pioglitazone and anti-inflammatory pharmaceuticals more broadly have potential as novel therapeutics for FASD.

Keywords: Brain; EtOH; Fetal Alcohol Spectrum Disorder; Microglia; Neuroinflammation.

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Figures

Fig. 1

Effect of ethanol and pioglitazone on IL-1β (panel A), TNF-α (panel B), and CCL2 (panel C) expression in neonatal mouse hippocampus. Animals were given 4 g/kg/day ethanol (E) daily by gavage on P4–9 and mice were sacrificed 1 day after the final dose of ethanol. Handled-only (H) and vehicle-treated (V) mice were included as controls. Where indicated, animals were treated with ethanol plus 12.5 mg/kg/day pioglitazone (P+E). The hippocampus was dissected, RNA prepared, cDNA synthesized, and mRNA levels were evaluated by real-time PCR. Results were normalized against β-actin and are expressed as fold changes relative to vehicle-treated control mice. Values are mean +/− SEM. PCR reactions were performed in duplicate on each sample. _n_=6–8 per treatment group. *** p < 0.001, ** p < 0.01 and * p < 0.05.

Fig. 2

Effect of ethanol and pioglitazone on IL-1β (panel A), TNF-α (panel B), and CCL2 (panel C) expression in neonatal mouse cerebellum. Animals were treated, cerebellum was dissected, and samples analyzed as described in Fig. 1. Results were normalized against β-actin and are expressed as fold changes relative to vehicle-treated control mice. Values are mean +/− SEM. *** p < 0.001, ** p < 0.01 and * p < 0.05.

Fig. 3

Effect of ethanol and pioglitazone on IL-1β (panel A), TNF-α (panel B), and CCL2 (panel C) expression in neonatal mouse cerebral cortex. Animals were treated, cerebral cortex was dissected, and samples analyzed as described in Fig. 1. Results were normalized against β-actin and are expressed as fold changes relative to vehicle-treated control mice. Values are mean +/− SEM. *** p < 0.001, ** p < 0.01 and * p < 0.05.

Fig. 4

Microglial morphology in neonatal mice treated with ethanol and pioglitazone. Animals were treated as described in Fig. 1. Photomicrographs illustrate the appearance of Iba-1 stained microglia in H, V, E, and P+E treated neonatal mice in the CA1 region of the hippocampus (panels A, D, G and J), lobule V of the cerebellar cortex (panels B, E, H and K), and the parietal region of the cerebral cortex (panels C, F, I and L). Handled animals = panels A–C. Vehicle animals = panels D–F. Ethanol-treated animals= panels G–I. P+E treated animals = panels J–L. Scale bar = 50 μm.

Fig. 5

Quantitative morphometric analysis of microglial morphology in neonatal mice treated with ethanol and pioglitazone. Animals were treated as described in Fig. 1 and processed for Iba-1 immunohistochemistry as in Fig. 4. Iba-1 stained microglia from H, V, E, and P+E treated animals were compared for relative cell area (panels A–C) and relative cell territory (panels D–F). A and D: hippocampus CA1 region. B and E: cerebellum lobule V. C and F: parietal cortex. Results are expressed as fold changes relative to vehicle-treated control mice. Values are mean +/− SEM. _n_=3 per treatment group. *** p < 0.001, ** p < 0.01 and * p < 0.05.

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Pioglitazone blocks ethanol induction of microglial activation and immune responses in the hippocampus, cerebellum, and cerebral cortex in a mouse model of fetal alcohol spectrum disorders - PubMed (original) (raw)