Wilkins Lincoln H, Prendergast Mark A, Blanchard John, Holley Robert C, Chambers Elton R, Littleton John M
Department of Behavioral Sciences, University of Kentucky, Lexington, Kentucky 40536-0298, USA.
Alcohol Clin Exp Res. 2006 Oct;30(10):1768-80. doi: 10.1111/j.1530-0277.2006.00210.x.
Previous studies have shown that withdrawal from ethanol (EtOH) exposure induces neuronal damage, as indicated by propidium iodide (PI) uptake, in organotypic hippocampal slice cultures. This is prevented by MK801, suggesting that damage is "excitotoxic," resulting from activation of N-methyl-d-aspartate (NMDA) receptors by endogenous glutamate. To avoid reliance on a single indicator, and to enable assessment of recovery from the EtOH withdrawal (EWD) insult, we assessed changes in cell markers for neurons and glia, as well as cell division, following either EWD or NMDA challenge (as a positive control).
Organotypic cultures from postnatal day (PND) 8 rats were cultured for 5 days before exposure to EtOH (mean concentration approximately 65 mM) for 10 days before EWD. Cultures of the same "days in vitro" age (DIV16) were exposed to NMDA (200 microM) for 1 hour. Neuronal injury was visualized using PI and indices of neurons, glia, or cell division were measured at intervals up to 10 days following the neurotoxic insults. Each time point and measurement used separate slice cultures, and these were treated as separate experiments with paired controls. Regional neuronal content was assessed by neuronal nuclear protein (NeuN) and calbindin D28k (Calb), glial content by glial fibrillary acidic protein (GFAP), and cell division by bromodeoxyuridine (BrdU) incorporation, all measured immunohistochemically.
Chronic exposure to EtOH was associated with a dramatic reduction in BrdU incorporation in all regions of cultures. Propidium iodide fluorescence in the CA1 region was elevated significantly after EWD and more so after NMDA challenge. Reduced immunoreactivity (IR) of NeuN and Calb suggested that loss of neurons resulted from the EWD insult. Bromodeoxyuridine incorporation was initially depressed even further by EWD, but had returned to control levels after 3 days. In contrast, following NMDA insult, BrdU incorporation was significantly and persistently elevated above control levels after 3 days. Glial fibrillary acidic protein was reduced immediately after both EWD and NMDA challenge. Several days after EWD, expression of neuronal and glial markers, although variable, had generally returned to control levels. In contrast, NeuN IR remained significantly reduced after NMDA challenge.
In general, the use of additional markers supports data obtained with PI uptake alone and suggests that neurons (and glia) are lost from the culture following EWD or NMDA challenge. These cell markers recover several days after EWD, but it is unclear whether functional recovery accompanies these changes. If the dramatic effect of EtOH exposure and EWD on BrdU incorporation reflects reduced neuro- and gliogenesis, it is likely that this adversely affects long-term recovery from EWD. Finally, some markers showed significant and consistent changes after EWD, whereas others did not. This information may facilitate the use of this model in evaluation of potential medications that protect against and/or promote recovery from neurotoxicity.
先前的研究表明，在器官型海马切片培养物中，从乙醇（EtOH）暴露中撤离会诱导神经元损伤，如碘化丙啶（PI）摄取所示。MK801可预防这种情况，这表明损伤是“兴奋性毒性的”，是由内源性谷氨酸激活N-甲基-D-天冬氨酸（NMDA）受体所致。为了避免依赖单一指标，并能够评估从乙醇撤离（EWD）损伤中恢复的情况，我们评估了EWD或NMDA激发（作为阳性对照）后神经元和神经胶质细胞标志物以及细胞分裂的变化。
出生后第8天（PND8）大鼠的器官型培养物在暴露于EtOH（平均浓度约65 mM）10天前培养5天，然后进行EWD。相同“体外培养天数”（DIV16）的培养物暴露于NMDA（200 μM）1小时。使用PI观察神经元损伤，并在神经毒性损伤后长达10天的间隔时间测量神经元、神经胶质细胞或细胞分裂的指标。每个时间点和测量使用单独的切片培养物，并且将这些视为具有配对对照的单独实验。通过神经元核蛋白（NeuN）和钙结合蛋白D28k（Calb）评估区域神经元含量，通过胶质纤维酸性蛋白（GFAP）评估神经胶质细胞含量，通过溴脱氧尿苷（BrdU）掺入评估细胞分裂，所有这些均通过免疫组织化学测量。
长期暴露于EtOH与培养物所有区域中BrdU掺入的显著减少有关。EWD后CA1区域的碘化丙啶荧光显著升高，NMDA激发后更是如此。NeuN和Calb免疫反应性（IR）降低表明神经元丢失是由EWD损伤所致。EWD最初使BrdU掺入进一步降低，但3天后已恢复到对照水平。相比之下，NMDA损伤后，3天后BrdU掺入显著且持续高于对照水平。EWD和NMDA激发后，胶质纤维酸性蛋白立即减少。EWD几天后，神经元和神经胶质细胞标志物的表达虽然有所变化，但总体上已恢复到对照水平。相比之下，NMDA激发后NeuN IR仍显著降低。
总体而言，使用额外的标志物支持仅通过PI摄取获得的数据，并表明EWD或NMDA激发后培养物中的神经元（和神经胶质细胞）会丢失。这些细胞标志物在EWD几天后恢复，但尚不清楚功能恢复是否伴随这些变化。如果EtOH暴露和EWD对BrdU掺入的显著影响反映了神经发生和胶质发生减少，那么这可能会对EWD的长期恢复产生不利影响。最后，一些标志物在EWD后显示出显著且一致的变化，而其他标志物则没有。这些信息可能有助于在评估预防和/或促进从神经毒性中恢复的潜在药物时使用该模型。
