A Practical Approach, Second Edition=Ronald D. Ho.pdf

EXPERIMENTAL APPROACHES TO EVALUATE MECHANISMS OF DEVELOPMENTAL TOXICITY 45Stereological methods provide invaluable tools for estimation of cell number in various structures.Effects caused by perturbations can be measured precisely and efficiently to determine if anexposure affects the volume of a structure or if it also influences particle number. Stereologicalmethods provide quantitation of pathological states that can then be used in quantitative riskassessments. 271,276Investigations of ethanol-induced neuronal loss highlight the usefulness of stereology in toxicology.Investigators have documented that the most severe deficits in cell number may occur inthe neocortex, the hippocampus, or the cerebellum, depending on the stage of CNS developmentin which exposure to ethanol occurs. 264–267,277 Not only are distinct regions of the brain affecteddifferently by ethanol, but the timing and pattern of exposure plays a critical role in the finaloutcome of ethanol-induced cellular loss. 278,279 The neocortex is particularly sensitive to neuronalloss following a relatively low exposure (resulting in a peak blood ethanol concentration [BEC] of150 mg/dl which is roughly equivalent to a BEC in humans after imbibing three to five standarddrinks) during early developmental events, including neurogenesis and migration.Decreases in cell numbers can be caused by a decrease in proliferation or an increase in celldeath, and various studies have shown ethanol to be a potent inhibitor of cellular proliferation. Asingle dose of ethanol administered to female rats within 8 h after mating results in a dose-dependentretardation of cell division in the fertilized ova, which is sustained up to 42 h after the exposure. 280Within the cerebral cortex, ethanol-exposed rat fetuses generate 30% fewer neocortical neuronsbetween gestational day (GD) 12 and 19, the peak time of cortical neurogenesis in the rat. 281 When3H-thymidine incorporation into rat fetal brain and liver tissue after exposure to ethanol in uteroon GD 16 and 20 was compared, the brain tissue showed decreased incorporation, suggestingincreased susceptibility of proliferating neuronal and glial precursors as opposed to proliferatingliver cells. 282In-depth in vivo research on neocortical neurogenesis in the mouse model has been performed,relating functional data (cell cycle rates and migration) within an anatomical context. 289,290 Ethanolinducedeffects documented include a reduction in the proliferating cell population and an increasein the length of the cell cycle, both contributing to fewer numbers of neurons or glial cellsgenerated. 283–286 The development of a cumulative BrdU incorporation technique allowed determinationof the effect of moderate alcohol intake (peak BEC of 153 mg/dl) on the cell cycle lengthof the proliferating cells of the dorsal neocortices. 266,287 These studies have shown 30% increase incell cycle length (18 h compared with 11 h) during early neocortical neurogenesis (GD 13 to 16);however, the increase was not constant throughout neurogenesis. As normal neurogenesis proceeds,the cell cycling rate naturally becomes longer, whereas the ethanol-exposed cells showed the samecell cycling rate throughout cortical neurogenesis. No increase in pyknotic cells was detected,suggesting again that the cycling cell is the target. 288The postnatal period of neocortical synaptogenesis for the rat, which includes the brain growthspurt, is a highly sensitive period. In vitro and in vivo studies suggest that ethanol neurotoxicityduring this period may be orchestrated by mechanisms different from those governing the earlierperiod of neocortical neurogenesis. Recently, convincing evidence of increased cell death due topostnatal exposure to ethanol has been documented. The period of natural cell death for the cerebralcortex occurs between postnatal days (PD) 1 and 10 in the rat, with a peak on PD 7. 292 Ikonomidou 293showed that by blocking N-methyl-D-aspartate (NMDA) glutamate receptors and activating γ-aminobutyric acid (GABA) receptors, ethanol triggers widespread apoptosis (as great as 30 timesthe baseline rate) in the synaptogenesis period of many brain regions, including the hippocampus,thalamus, and frontal, parietal, cingulate, and retrosplenial cortex. In this study, cell death wasmeasured by DeOlmos silver staining and was confirmed with caspase 3 activation in a more recentstudy. 294 Furthermore, if blood concentrations exceeded 200 mg/dl for 4 h, apoptotic neurodegenerationwas significantly increased compared with controls. If this threshold was exceeded for morethan 4 h, the degenerative response became progressively more severe in proportion to the length© 2006 by Taylor & Francis Group, LLC