Microglia play a role in ethanol-induced oxidative stress and apoptosis in developing hypothalamic neurons.

Ethanol consumption affects normal fetal brain growth and reduces the number of neurons in the various brain regions, including thehippocampus, cerebral cortex, cerebellum, olfactory bulb, and hypothalamus (Chen et al., 2004; De et al; 1994; Goodlett et al., 1991; Miller, 1995; Miller and Potempa, 1990; West et al., 1984). Within the hypothalamus, prenatal ethanol has been shown to produce functional abnormalities of several neuronal populations including β-endorphin (Sarkar et al., 2007), CRH (Lee et al., 2000), α-MSH, NPY, galanin (Barson et al., 2010), orexin 1 (Stettner et al., 2011), arginine vasopressin (Bird et al., 2006), vasoactive intestinal peptide (Rojas et al., 1999) and luteinizing hormone releasing hormone producing neurons (Scott et al., 1995). In addition prenatal ethanol has been shown to produce alteration in the body’s clock regulatory mechanism within the suprachiasmatic nucleus of the hypothalamus (Chen et al., 2006). Many of the functional defects of the hypothalamus in prenatal ethanol-exposed animals are related to the loss of the neuronal cell population (Baker and Shoemaker, 1995; De et al., 1994; Sarkar et al., 2007) Oxidative stress has been proposed as a mechanism of ethanol teratogenicity (Cohen-Kerem and Koren, 2003). The formation of ROS, including oxygen free radicals, occurs intracellularly in various tissues of rodent species following ethanol administration (Reinke et al., 1987). In the rat, ethanol also can perturb intracellular antioxidant pathways, including glutathione (GSH), GSH peroxidase, superoxide dismutase and catalase (Schlorff et al., 1999). Free radicals can react chemically with key cell macromolecules, namely, phospholipids, proteins and DNA, which may lead to cell dysfunction and eventual cell demise. In the brain, ethanol can produce oxidative stress by direct and indirect mechanisms, which can enhance ROS production (Montoliu et al., 1995). Ethanol also can increase the sensitivity of the brain to oxidative stress by selective mitochondrial injury (Ramachandran et al., 2001), thus compromising antioxidant pathways such as GSH (Rathinam et al., 2006). Because heavy alcohol exposure causes oxidative stress in developing neurons (for a review see Cohen-Kerem and Koren, 2003), we hypothesized that rats exposed to ethanol in utero may have oxidative damage to the hypothalamus, altering the cell-cell communication between neurons and other cells including microglia and leading to the activation of apoptotic processes in the neuronal population in the hypothalamus. Oxidative stress, in which production of ROS overwhelms antioxidant defenses, is a feature of many neurological diseases and neurodegeneration (Halliwell, 2006; Lin and Beal, 2006). ROS generated extracellularly and intracellularly directly oxidize and damage macromolecules such as DNA, proteins, and lipids, culminating in neurodegeneration of the CNS. Neurons are most susceptible to direct oxidative injury by ROS, which can also indirectly contribute to tissue damage by activating a number of cellular pathways that lead to the expression of stress-sensitive genes and proteins that cause oxidative injury. Recently, we have shown that ethanol treatment increased the release of various cytokines such as TNF-α, IL-1β, IL-6 from microglial cells and that TNF-α caused apoptotic cell death of developing hypothalamic neurons in vitro (Boyadjieva and Sarkar, 2010). A number of studies have indicated that the microglia are a source of ROS in the brain (Chao et al., 1992; Colton and Gilbert, 1987). The production of ROS by microglia is a part of the immune defense in the brain. A limited number of studies focused on the role of ROS generated from microglia in apoptosis of neurons (Min et al., 2003; Wang et al., 2008), and also the role of ROS from ethanol-treated microglia in apoptosis of developing hypothalamic neurons is not studied. In this study, we determined whether ethanol-treated microglial cell culture generate ROS and increase apoptotic cell death of fetal hypothalamic neurons in culture.