Autoradiographic Study of Regional Protein Synthesis in Focal Cerebral Ischemia with TCA Wash and Image Subtraction Techniques

Abstract

The standard biochemical method of trichloracetic acid (TCA) wash and the image processing technique were combined to differentiate and visualize the distributions of polypeptide-incorporated and unincorporated tracers in an autoradiographic study of regional protein synthesis, The validity of applying TCA wash procedures to cryostat sections was considered by histologic and chemical evaluations, For the autoradiographic study of in vivo protein synthesis, a tracer dose of L-[¹⁴C]valine was administered 30 min after occlusion of the posterior communicating artery in gerbils. Images of total (polypeptide-incorporated and unincorporated) radioactivity and of polypeptide-incorporated radioactivity were obtained from an identical cryostat section before and after TCA wash. The polypeptide-unincorporated radioactivity image was produced with an image processing system by subtracting pixel by pixel the polypeptide-incorporated radioactivity from the total radioactivity. The present study clearly demonstrated that in spite of the sufficient delivery of tracer amino acids, the polypeptide synthesis was completely lost in the ischemic focus. Free tracer was markedly accumulated in the brain adjacent to the ischemic focus. This kind of autoradiographic technique seems to be indispensable in studying the topographical complexity of the altered protein metabolism in the pathologic brain.