A sequential double-label 14C- and 3H-2-DG technique: validation by double-dissociation of functional states

Abstract

We investigated a double-label 2-DG protocol and method of analysis in which sequential injections of ³H- and ¹⁴C-2-DG were used to map brain metabolism during two distinct experimental treatments in the same animal. In initial studies, brain sections from rats given only ³H-2-DG or only ¹⁴C-2-DG were exposed on Ultrofilm and on X-ray film with an interposed sheet of mylar (X-ray/mylar). These studies were needed to determine whether, at the 50 ∶ 1 ³H ∶ ¹⁴C dose ratio used, ³H-2-DG uptake would be revealed only in Ultrofilm images and ¹⁴C-2-DG uptake only in X-ray/mylar images. We found that X-ray/mylar images indeed showed only ¹⁴C-2-DG uptake as ³H emissions were blocked by the protective coating of the film and the mylar. By contrast, Ultrofilm autoradiograms showed the 2-DG uptake pattern for both the ¹⁴C-2-DG and ³H-2-DG cases. We then examined autoradiograms from double-label cases in which ¹⁴C-2-DG and ³H-2-DG were sequentially given using a 100 ∶ 1³H ∶ ¹⁴C dose ratio, with a different treatment following each injection. As predicted from the single-label cases, activity in the X-ray/mylar images corresponded to the treatment that followed the ¹⁴C-2-DG injection, while the Ultrofilm images reflected both treatments and thus were not veridical representations of ³H label. This paper provides a solution to the contamination of Ultrofilm by ¹⁴C label in that we devised a subtraction algorithm using a computerized imaging system which removes the contaminating ¹⁴C from the Ultrofilm image, leaving a ‘Difference’ image of ³H-2-DG uptake. Difference images revealed activity consistent with the treatment that followed the ³H-2-DG injection. Thus, the X-ray/mylar and difference images separately indexed metabolic activity for two different functional states in the same subject. By allowing a subject to serve as its own control, this double-label method greatly increases the applicability and power of the 2-DG method.