Unraveling the ischemic brain transcriptome in a permanent middle cerebral artery occlusion mouse model by DNA microarray analysis

Abstract

Brain ischemia, also termed cerebral ischemia, is a condition in which there is insufficient blood flow to the brain to meet metabolic demand, leading to 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, as well as the underlying mechanisms of their action. The present study was initiated to investigate molecular responses at the level of gene expression in ischemic brain tissue. To achieve this, we used a mouse permanent middle cerebral artery occlusion (PMCAO) model in combination with high-throughput DNA microarray analysis on an Agilent microarray platform. Briefly, the right (ipsilateral) and left (contralateral) hemispheres of PMCAO model mice were dissected at two time points, 6 and 24 hours post-ischemia. Total RNA from the ischemic (ipsilateral) hemisphere was subjected to DNA microarray analysis on a mouse whole genome 4x44K DNA chip using a dye-swap approach. Functional categorization using the gene ontology (GO, MGD/AMIGO) of numerous changed genes revealed expression pattern changes in the major categories of cellular process, biological regulation, regulation of biological process, metabolic process and response to stimulus. Reverse-transcriptase PCR (RT-PCR) analysis on randomly selected highly up- or downregulated genes validated, in general, the microarray data. Using two time points for this analysis, major and minor trends in gene expression and/or functions were observed in relation to early- and late-response genes and differentially regulated genes that were further classified into specific pathways or disease states. We also examined the expression of these genes in the contralateral hemisphere, which suggested the presence of bilateral effects and/or differential regulation. This study provides the first ischemia-related transcriptome analysis of the mouse brain, laying a strong foundation for studies designed to elucidate the mechanisms regulating ischemia and to explore the neuroprotective effects of agents such as target neuropeptides.

Fig. 1.

Workflow from sampling and grinding of the brain hemisphere, total RNA extraction, and DNA microarray analysis of the ischemic brain (ipsilateral hemisphere). (A) Mouse whole brain showing the ipsilateral (right; boxed region) and contralateral (left) hemispheres. Brain tissues were ground to a fine powder in liquid nitrogen and stored at −80°C. (B) Total RNA extraction from the finely powdered brain tissues. Total RNA quality was confirmed by both agarose-gel electrophoresis and NanoDrop spectrophotometry. (C) DNA microarray chip showing the hybridized sample combinations (Sham × PMCAO at 6 and 24 hours) and dye-swap (Cy3 vs Cy5).

Fig. 2.

Differentially expressed genes in the ipsilateral hemisphere at 6 and 24 hours. The numbers above each bar indicate the selection of genes from the total microarray datasets within a defined fold range of greater than 1.5- or 2.0-fold and less than 0.75- or 0.5-fold. The gene lists are presented in supplementary material Tables S2–S7.

Fig. 3.

Functional categorization of the differentially expressed genes based on Gene Ontology (GO). The genes were grouped into the major categories of biological function, molecular function and cellular component. Numbers above each graph represent gene numbers for each subcategory.

Fig. 4.

See next page for legend.

Fig. 4.

Pathway- and disease-states-focused gene classification. The up- and downregulated genes at 6 (A) and 24 (B) hours after ischemia (ipsilateral hemisphere) were classified based on the available categories of more than 100 biological pathways or specific disease states in the SABiosciences PCR array list (QIAGEN; www.sabiosciences.com) for Mus musculus. The numbers in the y-axis represent number of genes in each category, which are indicated on the x-axis.

Fig. 5.

mRNA expression profiles of 16 differentially expressed genes. (A) Upregulated genes; (B) downregulated genes. Gel images on top show the PCR product bands stained with ethidium bromide; the band intensities are also presented graphically below for clarity. Lane numbers 1–8 indicate sham control (lanes 1, 3, 5 and 7) and PMCAO (lanes 2, 4, 6 and 8) treatment, respectively. RT-PCR was performed as described in the Methods and the primers are detailed in supplementary material Table S1.