Cheema Z F, West J R, Miranda R C
Department of Human Anatomy and Medical Neurobiology, Texas A&M University System Health Science Center, College Station 77843, USA.
Alcohol Clin Exp Res. 2000 Apr;24(4):535-43.
Animal studies modeling fetal alcohol syndrome have demonstrated that developmental exposure to alcohol is associated with decreased brain weight and significant neuronal loss in multiple regions of the developing brain. Our previous data suggest that the Fas/Apo [apoptosis]-1 receptor is transiently expressed in the developing cerebral cortex during the peak period of naturally occurring apoptotic cell death and maximum sensitivity to alcohol. Therefore, we hypothesized that ethanol increases the expression of suicide receptors such as Fas/Apo-1 in the developing fetal cerebral cortex and leads to an upregulation or extension of the normal period of apoptosis and consequent disorganization of the neural circuitry.
Ethanol was administered in one of four doses (120, 320, 630, and 950 mg/dl) to organotypic explant cultures of the developing cerebral cortex established from postnatal day 2 rats and maintained for 6 days in vitro. The number of cells expressing Fas/Apo-1 receptor mRNA was counted. Apoptosis was measured by the use of two independent assays; a cell death enzyme-linked immunosorbent assay for DNA fragmentation and flow cytometric analysis of Annexin-V binding to phosphatidylserine externalized to the outer leaflet of the plasma membrane. Necrosis was also estimated by two independent measures, the amount of lactate dehydrogenase released into culture medium and flow cytometric analysis of cells that were positive for both Annexin-V and propidium iodide.
A significantly larger number of developing cortical cells expressed Fas/Apo-1 mRNA at the lower doses (120 and 320 mg/dl) than at the higher doses (630 and 950 mg/dl). Furthermore, ethanol induced apoptosis in a dose-related manner, with peak apoptosis observed at a dose of 630 mg/dl in the case of DNA fragmentation and at 630 and 950 mg/dl in the case of phosphatidylserine translocation to the outer leaflet of the plasma membrane. Ethanol did not induce necrosis at any of the administered doses of ethanol.
Our data suggest that ethanol induces a susceptibility to apoptotic signals at low doses by upregulating the expression of mRNAs for cytotoxic receptors such as Fas/Apo-1 in the developing cerebral cortex. However, ethanol itself specifically induces apoptosis in the developing cerebral cortex only at higher doses.
模拟胎儿酒精综合征的动物研究表明，发育过程中暴露于酒精与脑重量减轻以及发育中大脑多个区域的显著神经元损失有关。我们之前的数据表明，Fas/Apo[凋亡]-1受体在自然发生的凋亡细胞死亡高峰期和对酒精的最大敏感性期间在发育中的大脑皮层中短暂表达。因此，我们推测乙醇会增加发育中的胎儿大脑皮层中诸如Fas/Apo-1等自杀受体的表达，并导致正常凋亡期的上调或延长，进而导致神经回路紊乱。
将乙醇以四种剂量（120、320、630和950mg/dl）之一给予从出生后第2天大鼠建立的发育中大脑皮层的器官型外植体培养物，并在体外维持6天。对表达Fas/Apo-1受体mRNA的细胞数量进行计数。通过两种独立的测定方法测量凋亡；一种用于DNA片段化的细胞死亡酶联免疫吸附测定以及膜联蛋白-V与质膜外小叶外化的磷脂酰丝氨酸结合的流式细胞术分析。坏死也通过两种独立的测量方法进行估计，释放到培养基中的乳酸脱氢酶的量以及对膜联蛋白-V和碘化丙啶均呈阳性的细胞的流式细胞术分析。
与较高剂量（630和950mg/dl）相比，较低剂量（120和320mg/dl）时，有显著更多的发育中的皮层细胞表达Fas/Apo-1mRNA。此外，乙醇以剂量相关的方式诱导凋亡，在DNA片段化的情况下，在630mg/dl的剂量下观察到凋亡峰值，在磷脂酰丝氨酸转运到质膜外小叶的情况下，在630和950mg/dl的剂量下观察到凋亡峰值。在任何给予的乙醇剂量下，乙醇均未诱导坏死。
我们的数据表明，乙醇通过上调发育中的大脑皮层中细胞毒性受体如Fas/Apo-1的mRNA表达，在低剂量时诱导对凋亡信号的敏感性。然而，乙醇本身仅在较高剂量时才特异性地诱导发育中的大脑皮层凋亡。
