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. 2017 Jul 24;14(1):145.
doi: 10.1186/s12974-017-0918-2.

TLR4 response mediates ethanol-induced neurodevelopment alterations in a model of fetal alcohol spectrum disorders

Affiliations

TLR4 response mediates ethanol-induced neurodevelopment alterations in a model of fetal alcohol spectrum disorders

María Pascual et al. J Neuroinflammation. .

Abstract

Background: Inflammation during brain development participates in the pathogenesis of early brain injury and cognitive dysfunctions. Prenatal ethanol exposure affects the developing brain and causes neural impairment, cognitive and behavioral effects, collectively known as fetal alcohol spectrum disorders (FASD). Our previous studies demonstrate that ethanol activates the innate immune response and TLR4 receptor and causes neuroinflammation, brain damage, and cognitive defects in the developmental brain stage of adolescents. We hypothesize that by activating the TLR4 response, maternal alcohol consumption during pregnancy triggers the release of cytokines and chemokines in both the maternal sera and brains of fetuses/offspring, which impairs brain ontogeny and causes cognitive dysfunction.

Methods: WT and TLR4-KO female mice treated with or without 10% ethanol in the drinking water during gestation and lactation were used. Cytokine/chemokine levels were determined by ELISA in the amniotic fluid, maternal serum, and cerebral cortex, as well as in the offspring cerebral cortex. Microglial and neuronal markers (evaluated by western blotting), myelin proteins (immunohistochemical and western blotting) and synaptic parameters (western blotting and electron microscopy) were assessed in the cortices of the WT and TLR4-KO pups on PND 0, 20, and 66. Behavioral tests (elevated plus maze and passive avoidance) were performed in the WT and TLR4-KO mice on PND 66 exposed or not to ethanol.

Results: We show that alcohol intake during gestation and lactation increases the levels of several cytokines/chemokines (IL-1β, IL-17, MIP-1α, and fractalkine) in the maternal sera, amniotic fluid, and brains of fetuses and offspring. The upregulation of cytokines/chemokines is associated with an increase in activated microglia markers (CD11b and MHC-II), and with a reduction in some synaptic (synaptotagmin, synapsin IIa) and myelin (MBP, PLP) proteins in the brains of offspring on days 0, 20, and 66 (long-term effects). These changes are associated with long-term behavioral impairments, in the 66-day-old alcohol-exposed pups. TLR4-deficient mice are protected against ethanol-induced cytokine/chemokine production in alcohol-treated dams and offspring, along with synaptic and myelin alterations, and the log-term behavioral dysfunction induced by ethanol in offspring.

Conclusions: These results suggest that the immune system activation, through the TLR4 response, might play an important role in the neurodevelopmental defects in FASD.

Keywords: Amniotic fluid; Behavior impairments; Cerebral cortex; Fetal alcohol spectrum disorders; Neuroinflammation; Prenatal ethanol exposure; Serum; TLR4.

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Conflict of interest statement

Ethics approval

All the experimental procedures were approved by the Ethical Committee of Animal Experimentation of the Príncipe Felipe Research Center (Valencia, Spain) and were carried out in accordance with the guidelines approved by the European Communities Council Directive (2010/63/EU) and Spanish Royal Decree 1201/2005.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Role of TLR4 in the expression of cytokines (IL-1β, IL-17) and chemokines (fractalkine, MCP-1, MIP-1α) in (a) maternal serum and amniotic fluid on E15, (b) in the maternal cerebral cortices of the WT and TLR4-KO pregnant female dams which underwent, or did not, chronic ethanol consumption, and (c) in the cerebral cortices of the WT and TLR4-KO embryos and pups on E15 and PND 0, 20, and 66 exposed, or not, to ethanol during the embryonic and postnatal periods. PPEE prenatal and postnatal ethanol exposure. Data represent mean ± SEM, n = 8 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001, compared to their respective control group, $p < 0.05, $$p < 0.01 compared to their corresponding control WT group
Fig. 2
Fig. 2
Role of TLR4 in the expression of several proteins induced by prenatal and postnatal ethanol exposure. Data represent the immunoblot analysis of PLP, MBP, synaptotagmin, synapsin IIa, Tuj-1, CD11b, MHC-II, caspase 3 (active fragment of 17 kDa), and the ELISA analysis of MAP-2 from the cerebral cortices of the WT and TLR4-KO pups on PND 0, 20, and 66 exposed (E), or not (C), to ethanol during the embryonic and postnatal periods. A representative immunoblot of each protein is shown. PPEE prenatal and postnatal ethanol exposure. Data represent mean ± SEM, n = 8 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001, compared to their respective control group
Fig. 3
Fig. 3
Role of TLR4 in prenatal and postnatal ethanol exposure in PLP, MBP, and Iba-1 immunoreactivity in the cortices of the WT and TLR4-KO pups. Arrows show examples of microglial activation. The scale bar is 50 μm. Bars represent the quantification values of PLP, MBP, and Iba-1 immunoreactivity, expressed as the thresholded area occupied by specific staining in the cortices of the WT and TLR4-KO mice treated, or not, prenatally and postnatally with ethanol (PPEE). Results are given as means ± SEM (n = 5). p < 0.05 compared to the ethanol-treated WT or TLR4-KO mice with their respective untreated control groups
Fig. 4
Fig. 4
The electron microscopy analysis shows the role of TLR4 in the synaptic structure alteration in the cerebral cortices of the pups (PND 20) exposed to ethanol. The representative transmission electron micrographs from the cortices of the WT and TLR4-KO pups on PND 20 exposed, or not, to ethanol during the prenatal and postnatal periods are shown. Arrows mark vesicles. PPEE prenatal and postnatal ethanol exposure. Scale bar represents 250 nm. Bars represent the vesicle number and postsynaptic density thickness. Data represent mean ± SEM, n = 5 pups/group. *p < 0.05, compared to their respective control group
Fig. 5
Fig. 5
Role of TLR4 in the passive avoidance test in the WT and TLR4-KO mice on PND 66 exposed, or not, to ethanol during the gestational and postnatal periods. Bars represent the time taken to enter the dark compartment during the passive avoidance test on the training day and during the test sessions (24 and 72 h after training). PPEE prenatal and postnatal ethanol exposure. Data are presented as mean (±SEM), n = 15 mice/group. +++p < 0.001, compared with the control WT mice during the training session; ###p < 0.001, compared with the respective group on the training day; *p > 0.05, **p < 0.01, compared with the ethanol pre-treated WT mice during the training session or the 24-h test; $p < 0.05, compared with their corresponding control pretreated group of mice on the 24-h test day

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