DNA Microarray Analysis Approach for Unraveling the Ischemic Mouse Brain Transcriptome

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. 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.