Early Delineation of Ischemic Tissue in Rat Brain Cryosections by High-Contrast Staining

Abstract

Background and Purpose —After short periods of ischemia, commonly used staining methods yield only moderate differences in optical contrast between normal and damaged brain tissue when gray-scale images are used for computer-assisted image analysis. We describe a high-contrast silver infarct staining (SIS) method that allows an early delineation of ischemic tissue as soon as 2 hours after middle cerebral artery occlusion (MCAO) in rat brain cryosections. Methods —Rats were subjected to permanent MCAO for 2, 4, 6, and 48 hours. The optical densities were quantified in nonischemic white and gray matter and in damaged tissue from gray-scale images of serial sections with the use of a video camera–based image analyzing system. SIS, hematoxylin-eosin, Nissl, and nitroblue tetrazolium stainings were performed in cryosections, and 2,3,5-triphenyltetrazolium hydrochloride (TTC) staining was performed in unfrozen vibratome sections. In addition, the range of reduced cerebral blood flow (CBF) in areas demarcated by SIS was determined in iodo[ 14 C]antipyrine autoradiograms of adjacent cryosections. Results —At all times after MCAO, only SIS showed significantly ( P <0.01) lower optical densities in damaged than in normal brain tissue for both white and gray matter. TTC staining was as effective as SIS 6 and 48 hours after MCAO. The tightest correlation between areas of reduced SIS and of reduced CBF was found at a mean ischemic CBF of 22.3 mL/100 g per minute. This corresponds to a CBF range of 0 to 44 mL/100 g per minute in areas of reduced SIS. Conclusions —In contrast to other staining methods, SIS allows a reliable delineation of ischemic brain tissue (core plus penumbra) from nonischemic white and gray matter of rat brain cryosections as soon as 2 hours after MCAO.