Shih C L, Chi S I, Chiu T H, Sun G Y, Lin T N
Institute of Medical Sciences, Tzu Chi College of Medicine and Humanities, Hualien, Taiwan.
Alcohol Clin Exp Res. 2001 Apr;25(4):612-8.
Although alcohol abusers are known to have higher incidences of hemorrhagic cerebrovascular diseases, it is not known whether these changes are associated with ethanol (EtOH) action on nitric oxide (NO) production in the cerebrovascular cells. The purpose of this study was to examine the effects of EtOH treatment on basal and cytokine-induced NO production in cortical pial cultures.
Cell cultures for this study included murine primary pial vascular cells, primary glial cells and cortical neurons. These cells were exposed to cytokines or EtOH for 24 to 48 hr. The culture media were used for measurement of nitrite, as an indication for NO release, and lactate dehydrogenase (LDH), as an index of cell membrane integrity. In addition, immunocytochemical determinations were carried out to identify cell types and to assess inducible nitric oxide synthase (iNOS).
Exposure of primary pial vascular cultures to cytokines that consisted of interleukin-1 beta (IL-1 beta; 250 pg/mL) and interferon-gamma (IFNgamma; 2 ng/mL) or to EtOH (50 to 100 mM) for 24 to 48 hr significantly elevated NO production. NO production could be attenuated by N-nitro-L-arginine (N-arg), a nonspecific NOS inhibitor, or aminoguanidine (AG), an iNOS inhibitor. Increased iNOS immunoreactivity was observed in cytokines- or EtOH-treated pial cells. When pial cells were cocultured with cortical neurons, prolonged EtOH exposure led to a large increase in NO production as well as LDH release. However, this increase was not observed in pial culture alone or in mixed cortical culture. Nevertheless, inhibition of NO production with N-arg or AG did not alter the EtOH-induced LDH release in the pial cells cocultured with cortical neurons.
These results show that EtOH exposure led to increased production of NO in primary pial cell culture. In mixed culture that contained cortical neurons and pial cells, EtOH induced increase in NO as well as LDH release, which is an indication of loss of cell membrane integrity. However, EtOH-mediated LDH release in mixed cortical pial cultures was not a consequence of the increase in NO production by these cells. Studies that use mixed cortical-pial cultures may provide a unique in vitro system for examining the interactions among glial cells, neurons, and cerebrovascular cells.
尽管已知酗酒者出血性脑血管疾病的发病率较高，但尚不清楚这些变化是否与乙醇（EtOH）对脑血管细胞中一氧化氮（NO）生成的作用有关。本研究的目的是检测EtOH处理对皮质软脑膜培养物中基础和细胞因子诱导的NO生成的影响。
本研究的细胞培养包括小鼠原代软脑膜血管细胞、原代神经胶质细胞和皮质神经元。将这些细胞暴露于细胞因子或EtOH中24至48小时。使用培养基测量亚硝酸盐，作为NO释放的指标，以及乳酸脱氢酶（LDH），作为细胞膜完整性的指标。此外，进行免疫细胞化学测定以鉴定细胞类型并评估诱导型一氧化氮合酶（iNOS）。
将原代软脑膜血管培养物暴露于由白细胞介素-1β（IL-1β；250 pg/mL）和干扰素-γ（IFNγ；2 ng/mL）组成的细胞因子或EtOH（50至100 mM）中24至48小时，可显著提高NO生成。非特异性NOS抑制剂N-硝基-L-精氨酸（N-arg）或iNOS抑制剂氨基胍（AG）可减弱NO生成。在细胞因子或EtOH处理的软脑膜细胞中观察到iNOS免疫反应性增加。当软脑膜细胞与皮质神经元共培养时，长时间暴露于EtOH会导致NO生成以及LDH释放大幅增加。然而，单独的软脑膜培养或混合皮质培养中未观察到这种增加。尽管如此，用N-arg或AG抑制NO生成并未改变与皮质神经元共培养的软脑膜细胞中EtOH诱导的LDH释放。
这些结果表明，暴露于EtOH会导致原代软脑膜细胞培养物中NO生成增加。在包含皮质神经元和软脑膜细胞的混合培养中，EtOH诱导NO以及LDH释放增加，这表明细胞膜完整性丧失。然而，EtOH介导的混合皮质软脑膜培养物中LDH释放并非这些细胞中NO生成增加的结果。使用混合皮质-软脑膜培养的研究可能为研究神经胶质细胞、神经元和脑血管细胞之间的相互作用提供一个独特的体外系统。
