Ethanol Regulates Angiogenic Cytokines During Neural Development: Evidence From an in Vitro Model of Mitogen‐Withdrawal–Induced Cerebral Cortical Neuroepithelial Differentiation

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Abstract
Background: Heavy alcohol consumption during pregnancy can cause significant mental retardation and brain damage. We recently showed that ethanol depletes reserve cerebral cortical stem cell capacity. Moreover, proliferating neuroepithelial cells exposed to ethanol were resistant to subsequent retinoic acid‐induced differentiation. Emerging evidence suggests that cytokines play a crucial growth‐promoting role in the developing neural tube. Methods: We cultured murine cortical neurosphere cultures in control or ethanol‐supplemented mitogenic medium, to mimic alcohol exposure during the period of neuroepithelial proliferation. Cultures were then treated with a step‐wise mitogen‐withdrawal, integrin‐activation model to mimic subsequent phases of neuronal migration and early differentiation. We examined the impact of alcohol exposure during neurogenesis on the secretion of inflammatory and growth‐promoting cytokines. Results: Cortical neurosphere cultures exhibit increasingly complex differentiation phenotypes in response to step‐wise mitogen‐withdrawal and laminin exposure. Some inflammation‐modulating cytokines were secreted independent of differentiation state. However, chemotactic cytokines were specifically secreted at high levels, as a function of differentiation stage. monocyte chemotactic protein‐1, vascular endothelial growth factor‐A, and interleukin (IL)‐10 were coordinately decreased during differentiation compared with neuroepithelial proliferation, while granulocyte macrophage‐colony stimulating factor (GM‐CSF) was induced during differentiation, compared with the neuroepithelial proliferation period. Ethanol exposure during the period of neuroepithelial proliferation prevented the early differentiation‐induced increase in GM‐CSF while inducing differentiation‐associated increase in IL‐12 secretion. Conclusion: Embryonic cerebral cortical neuroepithelial‐derived precursors secrete high levels of several angiogenic and neural‐growth–promoting cytokines as they differentiate into neurons. Our data collectively suggest that ethanol exposure during the period of neuroepithelial proliferation significantly disrupts cytokine signals that are required for the support of emerging neurovascular networks, and the maintenance of neural stem cell beds.