Pixel-based image analysis of HSP70, GADD45 and MAP2 mRNA expression after focal cerebral ischemia: hemodynamic and histological correlates

Gene expression studies with in situ hybridization after focal brain ischemia indicate a variety of distinct anatomical patterns. An important question is to what extent such reactive gene expression correlates with neuronal damage or survival. To study these questions, we focused on two stressed-induced genes, heat shock protein 70 (HSP70) and growth-arrest and DNA damage-inducible gene (GADD) 45 mRNA, and we compared reactive changes in mRNA to loss of the constitutive signal for microtubule-associated protein 2 (MAP2) mRNA. A pixel-based image analysis of mRNA signals was carried out using a highly reproducible model of focal brain ischemia. A poly-l-lysine coated filament was used to occlude the origin of the middle cerebral artery (MCA) for 2 h in ventilated, normothermic rats. Brains were collected after 0, 1, 3 and 6 h, and 1, 3 and 7 days. In situ hybridization analysis was carried out for HSP70 mRNA, GADD45 mRNA and MAP2 mRNA. Autoradiographic data sets were averaged and co-mapped into a common template of the rat brain. These data sets were then compared on a pixel-by-pixel basis with previously acquired image data sets derived from quantitative studies of local cerebral blood flow (LCBF) (obtained at the end of 2-h ischemia) of and infarctive histopathology (obtained at 3 days) in the same focal ischemia model. HSP70 mRNA and GADD45 mRNA were grossly elevated in the hemisphere subjected to ischemia during the first day. Pixel-based analysis showed a strong correlation between HSP70 mRNA signals, the degree of early blood-flow reduction and the probability of histological infarction. GADD45 mRNA was expressed in a more variable fashion. Decreases in MAP2 mRNA signals at 1, 3 and 7 days correlated strongly with histological infarction. These co-mapping procedures allow us to conclude that HSP70 mRNA is a robust indicator of ischemic stress and histological outcome after 2 h of focal brain ischemia. The topographic features of GADD45 expression suggest its possible role in conferring resistance to ischemic injury. Finally, our results indicate that local decreases in constitutive MAP2 expression at 1 day and beyond may be used as a robust marker of tissue regions having a high probability of focal infarction.