Identification of differentially expressed genes in rat hippocampus after transient global cerebral ischemia using subtractive cDNA cloning based on polymerase chain reaction

Background and Purpose-The purpose of this study is to identify new molecul es that play important roles in the phenomena that occur in the hippocampus after transient global cerebral ischemia, as clues to better understanding of the mechanisms. Methods-A subtractive cDNA library was established by suppression subtracti ve hybridization of rat hippocampal tissues after transient global cerebral ischemia. With differential screening of the library, upregulated fragment s were identified. The mRNA expression levels of selected genes were measur ed with semiquantitative reverse transcriptase polymerase chain reaction (P CR). Results-Among more than 100 isolated fragments, approximately half were det ermined to be identical to known sequences. The rest showed high homology t o known sequences, and only 2 did not exhibit homology to any known sequenc es. The expression of 5 genes identified in this study increased in 24 hour s after ischemia to a level twice as high as that in sham-operated controls . These included furin, prosaposin, synaptotagmin IV, heat shock protein 10 5, and the neutral and basic amino acid transporter (NBAT). The increases i n the mRNA expression levels of the genes except NBAT, as revealed by semiq uantitative reverse transcription PCR, were statistically significant at bo th 6 and 24 hours after ischemia. Conclusions-Genes isolated are thought to be associated with production of proteins necessary for degeneration, neuroprotection, and reconstruction of neurons. How the expression of these genes relates to functional changes a fter ischemia remains to be determined. PCR-based subtractive cDNA cloning is demonstrated to be a useful tool for analyzing in vivo gene expression i n animal ischemia models.