Multiple oxygen tension environments reveal diverse patterns of transcriptional regulation in primary astrocytes. Rattus norvegicus

The central nervous system normally functions at O2 levels which would be regarded as hypoxic by most other tissues. However, most in vitro studies of neurons and astrocytes are conducted under hyperoxic conditions without consideration of O2-dependent cellular adaptation. We analyzed the reactivity of astrocytes to 1, 4 and 9% O2 tensions compared to the cell culture standard of 20% O2, to investigate their ability to sense and translate this O2 information to transcriptional activity. Variance of ambient O2 tension for rat astrocytes resulted in profound changes in ribosomal activity, cytoskeletal and energy-regulatory mechanisms and cytokine-related signaling. Clustering of transcriptional regulation patterns revealed four distinct response pattern groups that directionally pivoted around the 4% O2 tension, or demonstrated coherent ascending/decreasing gene expression patterns in response to diverse oxygen tensions. Immune response and cell cycle/cancer-related signaling pathway transcriptomic subsets were significantly activated with increasing hypoxia, whilst hemostatic and cardiovascular signaling mechanisms were attenuated with increasing hypoxia. Our data indicate that variant O2 tensions induce specific and physiologically-focused transcript regulation patterns that may underpin important physiological mechanisms that connect higher neurological activity to astrocytic function and ambient oxygen environments. These strongly defined patterns demonstrate a strong bias for physiological transcript programs to pivot around the 4% O2 tension, while uni-modal programs that do not, appear more related to pathological actions. The functional interaction of these transcriptional ‘programs’ may serve to regulate the dynamic vascular responsivity of the central nervous system during periods of stress or heightened activity. Overall design: Cerebral cortices were removed from 6-8-day-old Wistar rats, cortical astrocytes were isolated and these cells were maintained in a humidified incubator at 37°C (95% air; 5% CO2). This resulted in a culture of cortical astrocytes, as confirmed by visual inspection the following day and later by glial fibrillary acidic protein immunohistochemistry. Any cortical astrocyte culture that was not homogenous was disposed of and not used in this study. Culture medium was exchanged every 7 days and cells were grown in culture for up to 14 days. 24hr prior to experimentation cells were transferred to an hypoxic workstation equilibrated with either 1%, 4%, 9% or 21% O2 and 5% CO2, with the remaining percentage gas being N2. Once cortical astrocytes had reached approximately 90% confluence (75cm2 flask) they were subjected to hypoxia as above, washed with PBS, removed from the flask base with 0.05% trypsin-EDTA (Gibco) and then gently centrifuged (500xg). The cell pellet was re-suspended in PBS, centrifuged twice more to remove any traces of media, then triturated in 8-10 volumes of RNAlater (Applied Biosystems), frozen and stored at -80oC. Messenger RNA was amplified and labeled with biotin using the standard Illumina protocol (Illumina TotalPrep RNA Amplification Kit Ambion; Austin, TX, cat # IL1791), and hybridized to Illumina's Sentrix Rat Ref-12,v1 Expression BeadChips (Illumina, San Diego, CA). Three replicates from each treatment group (1%, 21%, 4%, and 9% O2) were hybridized and the data was extracted using the Illumina BeadStudio software(v3.4).

中枢神经系统（central nervous system）的正常运作所依赖的氧分压，在多数其他组织中会被判定为缺氧状态。然而，绝大多数针对神经元与星形胶质细胞（astrocytes）的体外研究均在高氧条件下开展，未考虑氧依赖的细胞适应机制。本研究以细胞培养标准氧分压（20% O2）为对照，分析星形胶质细胞对1%、4%及9%氧分压的反应性，以探究其感知氧分压信息并将其转化为转录活性的能力。

大鼠星形胶质细胞所处环境氧分压的变化，会对核糖体活性、细胞骨架与能量调控机制以及细胞因子相关信号通路产生显著影响。转录调控模式的聚类分析显示，存在4种截然不同的反应模式组：要么以4%氧分压为方向拐点，要么在应对不同氧分压时呈现出一致的基因表达上调/下调趋势。随着缺氧程度加剧，免疫应答以及细胞周期/癌症相关信号通路的转录组子集显著激活；而止血与心血管信号调控机制则随缺氧程度加重被抑制。

本研究数据表明，不同氧分压会诱导出具有特异性且以生理功能为导向的转录调控模式，这类模式可能是连接高级神经活动、星形胶质细胞功能与环境氧分压的重要生理机制的基础。这些特征明确的模式显示，生理相关转录程序大多以4%氧分压为拐点；而不具备该特征的单峰转录程序，则更多与病理过程相关。这些转录‘程序’之间的功能互作，可能在机体应激或神经活动增强时，调控中枢神经系统的动态血管反应性。

整体实验设计：从6~8日龄的Wistar大鼠体内取出大脑皮层，分离皮层星形胶质细胞，将细胞置于37℃、湿度饱和的培养箱中培养（95%空气；5% CO2）。次日通过目视检查，后续通过胶质纤维酸性蛋白（glial fibrillary acidic protein, GFAP）免疫组织化学验证，确认为皮层星形胶质细胞纯培养物。若培养物不纯则弃用，不纳入本研究。培养液每7天更换一次，细胞最长培养至14天。实验前24小时，将细胞转移至缺氧工作站（hypoxic workstation），分别用1%、4%、9%或21% O2与5% CO2平衡气体（其余气体为氮气）进行培养。待皮层星形胶质细胞在75cm²培养瓶中汇合度达到约90%时，按照前述条件进行缺氧处理：先用磷酸盐缓冲液（phosphate buffered saline, PBS）洗涤细胞，再用0.05%胰蛋白酶-乙二胺四乙酸（trypsin-ethylenediaminetetraacetic acid, trypsin-EDTA，Gibco）消化贴壁细胞，随后以500×g的转速轻轻离心。将细胞沉淀重悬于PBS中，再离心两次以去除残留培养基，之后用8~10倍体积的RNAlater（Applied Biosystems）重悬细胞，冻存并保存在-80℃环境中。

采用标准Illumina总RNA扩增方案（Illumina TotalPrep RNA Amplification Kit，Ambion，美国德克萨斯州奥斯汀，货号IL1791）对信使RNA进行扩增并以生物素标记，随后将标记产物与Illumina Sentrix Rat Ref-12,v1 Expression BeadChips（Illumina，美国加利福尼亚州圣地亚哥）进行杂交。每个处理组（1%、21%、4%、9% O2）设置3个生物学重复，使用Illumina BeadStudio软件（v3.4）提取数据。