DNA Microarray Analysis Approach for Unraveling the Ischemic Mouse Brain Transcriptome. Mus musculus

Brain ischemia, also termed cerebral ischemia, is a condition in which there is insufficient blood flow to the brain to meet metabolic demand, and results in brain tissue death (cerebral infarction) due to poor oxygen supply (cerebral hypoxia). Our group is interested in the protective effects of neuropeptides for alleviating brain ischemia, and mechanisms therein. However, before proceeding to the neuroprotection aspect of our research, we initiated a study with a primary aim to investigate the molecular responses at the level of gene expression in ischemic brain tissue. To do so, we used permanent middle cerebral artery occlusion (PMCAO) model mice in combination with high-throughput DNA microarray analysis on an Agilent microarray platform. Briefly, saline injected mice brain in sham (control, n=3) and PMCAO (treatment, n=3) mice were dissected into left (contralateral) and right hemispheres (ipsilateral) at two time points, 6 and 24 h after injection. The ipsilateral hemisphere shows ischemia, within 24 h, and is marked by cell death as visualized by TTC staining. Tissues were ground into fine powder with liquid nitrogen and total RNA was extracted, followed by quality check using gel electrophoresis and cDNA synthesis in conjunction with RT-PCR using certain marker genes. The obtained good quality total RNA from ipsilateral hemisphere was used for DNA microarray analysis on a mouse (Mus musculus) whole genome 4x44K DNA chip by using the dye-swap approach. Results revealed a large number of changed gene expressions at both 6 (1237 up- and 620 down-regulated) and 24 h (2759 up- and 2102 down-regulated). 792 and 167 genes were found to be commonly up- and down-regulated 6 and 24 h post-ischemia, respectively. Functional categorization using the gene ontology (GO, MGD/AMIGO) of these gene expressions revealed major categories of cellular processes, biological regulation, regulation of biological processes, metabolic processes, and response to stimulus. In addition, RT-PCR using specific primers of randomly selected genes was used to validate the changed gene expressions. This study provides the first inventory of ischemia-related transcriptome in mouse brain. Overall design: For appropriate control to the PMCAO model, mice were anesthetized with 4% isoflurane (induction) and 2% isoflurane (maintenance) in a 30% O2 and 70% N2O gas mixture via a face mask. An incision was then made in the cervical skin followed by opening of salivary gland, and visualization of the right common carotid artery. The external carotid artery was exposed through a midline cervical incision, and subsequently, the wound was closed by sutures. For the PMCAO model, we used the intraluminal filament technique, where a 7-0 monofilament nylon suture with its tip slightly rounded by heating was inserted into the common carotid artery followed by placement into the middle cerebral artery. In both the control and PMCAO model, saline (0.9% NaCl) was injected into the intracerebroventricle, and the animals were returned to their cages. A total of eight groups were prepared with three mice each in the control (4 groups) and PMCAO model (4 groups). After 6 and 24 h post-injection of saline, mice were removed from the cages and decapitated using scissors, and the whole brains were carefully dissected on ice. The left (contralateral) and right (ipsilateral) hemispheres were separated and placed in 2 mL Eppendorf tubes followed by quickly immersing the tubes in liquid nitrogen (Lq. N2), and then stored in -80ºC prior to further analysis. A mouse 4 x 44K whole genome oligo DNA microarray chip (G4122F, Agilent Technologies, Palo Alto, CA, USA) was used for global gene expression analysis using the contralateral hemispheres. The effects of ischemia were checked over the SHAM control mice at 6 and 24 h post-saline injection.

脑缺血（cerebral ischemia）又称大脑缺血，是一种因脑部血流灌注不足无法满足代谢需求，进而因氧供匮乏（cerebral hypoxia，脑缺氧）导致脑组织死亡（cerebral infarction，脑梗死）的病症。本课题组聚焦于神经肽缓解脑缺血的保护作用及其内在机制。然而，在推进本研究的神经保护分支之前，我们率先开展了一项核心目标为探究缺血脑组织基因表达水平分子响应的研究。

为此，我们采用永久性大脑中动脉闭塞（PMCAO）模型小鼠，并结合安捷伦（Agilent）微阵列平台的高通量DNA微阵列分析技术。简言之，在生理盐水注射后的两个时间点（6小时与24小时），分别对假手术组（对照组，n=3）与PMCAO模型组（处理组，n=3）的小鼠脑组织进行解剖，分离为左侧（对侧）与右侧（同侧）大脑半球。同侧大脑半球在24小时内呈现缺血状态，并可通过TTC染色直观观察到细胞死亡现象。将组织用液氮研磨为细粉后提取总RNA，随后通过凝胶电泳进行质量检测，并结合特定标记基因的逆转录PCR（RT-PCR）进行cDNA合成。将从同侧半球获取的合格总RNA，采用荧光染料互换（dye-swap）方法，在小鼠（Mus musculus）全基因组4×44K DNA芯片上进行DNA微阵列分析。

分析结果显示，在缺血后6小时与24小时均存在大量基因表达差异：6小时组有1237个基因上调、620个基因下调，24小时组则有2759个基因上调、2102个基因下调。分别有792个与167个基因在缺血后6小时与24小时时呈现共同上调与共同下调。通过基因本体（GO, MGD/AMIGO）对这些差异表达基因进行功能注释分类，发现主要富集于细胞过程、生物调控、生物过程调控、代谢过程以及应激响应等类别。此外，我们采用针对随机筛选基因的特异性引物进行RT-PCR实验，对差异表达基因进行验证。本研究首次构建了小鼠脑内缺血相关转录组的完整图谱。

实验整体设计：为对PMCAO模型设置恰当对照，小鼠通过面罩吸入由30%氧气、70%一氧化二氮组成的混合气体，采用4%异氟烷进行诱导麻醉，2%异氟烷维持麻醉状态。随后于颈部皮肤做切口，分离唾液腺并暴露右侧颈总动脉；通过颈部正中切口暴露颈外动脉，随后用缝线缝合伤口。针对PMCAO模型，我们采用线栓法：将尖端经加热轻微圆润化的7-0单丝尼龙缝线插入颈总动脉，随后推送至大脑中动脉。对照组与PMCAO模型组小鼠均向脑室内注射生理盐水（0.9% NaCl），随后将动物放回饲养笼。实验共设置8组小鼠，每组3只：对照组4组，PMCAO模型组4组。在生理盐水注射后6小时与24小时时，将小鼠从饲养笼取出，用剪刀处死并断头，随后在冰面上小心解剖全脑。分离左侧（对侧）与右侧（同侧）大脑半球，置于2 mL离心管中后快速浸入液氮（Lq. N2），随后于-80℃冰箱保存以待后续分析。我们采用小鼠4×44K全基因组寡核苷酸DNA微阵列芯片（G4122F，安捷伦科技公司，美国加州帕洛阿尔托），通过对侧大脑半球样本进行全基因组基因表达分析。在生理盐水注射后6小时与24小时，通过假手术对照组小鼠样本验证缺血效应。