Simultaneous measurement of cerebral blood flow and mRNA signals: pixel-based inter-modality correlational analysis

The analysis of pixel-based relationships between local cerebral blood flow (LCBF) and mRNA expression can reveal important insights into brain functi on. Traditionally, LCBF and in situ hybridization studies for genes of inte rest have been analyzed in separate series. To overcome this limitation and to increase the power of statistical analysis, this study focused on devel oping a double-label method to measure local cerebral blood flow (LCBF) and gene expressions simultaneously by means of a dual-autoradiography procedu re. A C-14-iodoantipyrine autoradiographic LCBF study was first performed. Serial brain sections (12 in this study) were obtained at multiple coronal levels and were processed in the conventional manner to yield quantitative LCBF images. Two replicate sections at each bregma level were then used for in situ hybridization. To eliminate the C-14-iodoantipyrine from these sec tions, a chloroform-washout procedure was first performed. The sections wer e then processed for in situ hybridization autoradiography for the probes o f interest. This method was tested in Wistar rats subjected to 12 min of gl obal forebrain ischemia by two-vessel occlusion plus hypotension, followed by 2 or 6 h of reperfusion (n = 4-6 per group). LCBF and in situ hybridizat ion images for heat shock protein 70 (HSP70) were generated for each rat, a ligned by disparity analysis, and analyzed on a pixel-by-pixel basis. This method yielded detailed inter-modality correlation between LCBF and HSP70 m RNA expressions. The advantages of this method include reducing the number of experimental animals by one-half; and providing accurate pixel-based cor relations between different modalities in the same animals, thus enabling p aired statistical analyses. This method can be extended to permit correlati on of LCBF with the expression of multiple genes of interest. (C) 2001 Else vier Science B.V. All rights reserved.