Role of microglia in ethanol's apoptotic action on hypothalamic neuronal cells in primary cultures.
BACKGROUND
Microglia are the major inflammatory cells in the central nervous system and play a role in brain injuries as well as brain diseases. In this study, we determined the role of microglia in ethanol's apoptotic action on neuronal cells obtained from the mediobasal hypothalamus and maintained in primary cultures. We also tested the effect of cAMP, a signaling molecule critically involved in hypothalamic neuronal survival, on microglia-mediated ethanol's neurotoxic action.


METHODS
Ethanol's neurotoxic action was determined on enriched fetal mediobasal hypothalamic neuronal cells with or without microglia cells or ethanol-activated microglia-conditioned media. Ethanol's apoptotic action was determined using nucleosome assay. Microglia activation was determined using OX6 histochemistry and by measuring inflammatory cytokines secretion from microglia in cultures using enzyme-linked immunosorbent assay (ELISA). An immunoneutralization study was conducted to identify the role of a cytokine involved in ethanol's apoptotic action.


RESULTS
We show here that ethanol at a dose range of 50 and 100 mM induces neuronal death by an apoptotic process. Ethanol's ability to induce an apoptotic death of neurons is increased by the presence of ethanol-activated microglia-conditioned media. In the presence of ethanol, microglia showed elevated secretion of various inflammatory cytokines, of which TNF-α shows significant apoptotic action on mediobasal hypothalamic neuronal cells. Ethanol's neurotoxic action was completely prevented by cAMP. The cell-signaling molecule also prevented ethanol-activated microglial production of TNF-α. Immunoneutralization of TNF-α prevented the microglia-derived media's ability to induce neuronal death.


CONCLUSIONS
These results suggest that ethanol's apoptotic action on hypothalamic neuronal cells might be mediated via microglia, possibly via increased production of TNF-α. Furthermore, cAMP reduces TNF-α production from microglia to prevent ethanol's neurotoxic action.
Boyadjieva
Nadka I
NI
Endocrine Program, Department of Animal Sciences, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08901, USA.
Sarkar
Dipak K
DK
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D002492:Central Nervous System Depressants / Q000037:antagonists & inhibitors / Q000633:toxicity*; D003470:Culture Media; D017077:Culture Media, Conditioned; D000242:Cyclic AMP / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000431:Ethanol / Q000037:antagonists & inhibitors / Q000633:toxicity*; D005260:Female; D007033:Hypothalamus, Middle / Q000166:cytology* / Q000187:drug effects; D007150:Immunohistochemistry; D017628:Microglia / Q000502:physiology*; D009474:Neurons / Q000187:drug effects*; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000502:physiology
D002492:Central Nervous System Depressants; D003470:Culture Media; D017077:Culture Media, Conditioned; D016207:Cytokines; D014409:Tumor Necrosis Factor-alpha; D000431:Ethanol; D000242:Cyclic AMP