In many tissues, short ischaemic / hypoxic intervals can induce tolerance against damage when followed by long lasting ischemic / hypoxic events (ischemic preconditioning). In the heart, this phenomenon has been extensively examined and ischaemic preconditioning is on the threshold to clinical application. In the brain this phenomenon exists, however, mechanisms have remained enigmatic. The aim of our project is to establish in-vitro-models of preconditioning in CNS tissues, and to study preconditioning mechanisms in these systems on the cellular and molecular level. To this end we have established hypoxic and pharmacologic (3-NPA, ouabaine) preconditioning in pure neuronal cultures. We use systematic (for the detection of all genes differently expressed by preconditioning) and manipulative (for the detection of factors interferring with transactivation / transcription) strategies. Candidate genes include bcl2 and iNOS. As a screening approach we are currently establishing SAGE, a quantitative high throughput approach based on the generation of short sequence tags unique to each transcript and the subsequent concatenation of many tags into one molecule which is amplified by PCR and sequenced. We are using preconditioning for the characterization of endogenous protective or damaging genetic mechanisms (´good`versus `bad`genes) and hope to develop clinically applicable for the induction of tolerance.
PCR, quantitative PCR, northern blotting, SAGE (seriell analysis of gene expression), non-radioactive automatic sequencing, cloning techniques, chamber for induced ischaemia in cell cultures, animal preparation procedures
SFB-507, DFG Me1562/1-1, DFG-454/8-1