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

Gene expression studies with in situ hybridization after focal brain ischem ia indicate a variety of distinct anatomical patterns. An important questio n is to what extent such reactive gene expression correlates with neuronal damage or survival. To study these questions, we focused on two stressed-in duced 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 carri ed out for HSP70 mRNA, GADD45 mRNA and MAP2 mRNA. Autoradiographic data set s were averaged and co-mapped into a common template of the rat brain. Thes e data sets were then compared on a pixel-by-pixel basis with previously ac quired image data sets derived from quantitative studies of local cerebral blood flow (LCBF) (obtained at the end of 2-h ischemia) of and infarctive h istopathology (obtained at 3 days) in the same focal ischemia model. HSP70 mRNA and GADD45 mRNA were grossly elevated in the hemisphere subjected to i schemia during the first day. Pixel-based analysis showed a strong correlat ion between HSP70 mRNA signals, the degree of early blood-flow reduction an d 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 procedur es allow us to conclude that HSP70 mRNA is a robust indicator of ischemic s tress and histological outcome after 2 h of focal brain ischemia. The topog raphic features of GADD45 expression suggest its possible role in conferrin g resistance to ischemic injury. Finally, our results indicate that local d ecreases 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 infar ction. (C) 1998 Elsevier Science B.V. All rights reserved.