Triphenyltetrazolium chloride (TTC) was used to delineate ischaemic lesions in the rat brain at various times following middle cerebral artery occlusion. A comparison was made of TTC staining by immersion and perfusion techniques and conventional light microscopy. The lesions were quantified by measuring the ischaemic area at the sections corresponding to 7 mm in front of the AO line (atlas of Konig and Klippel). In animals examined 24 h after middle cerebral artery occlusion (MCAO), the area of infarction was 17.4 +/- 1.3 mm2 on the TTC perfused slices and 17.6 +/- 1.6 mm2 on the TTC immersed slices (mean +/- SEM). By contrast, there was a marked difference between the two TTC methods when tissues were examined at shorter intervals after artery occlusion. In the TTC-perfused animals, there was no significant difference between the mean areas of infarction measured at 5-20 min, 3-4 h, or 24 h post occlusion. Immersion in TTC, however, failed to reveal any consistent ischaemic damage when applied at the earlier post-occlusion times. Conventional histopathology demonstrated minimal lesions at 5-20 min but at 4 h or more the lesions were not significantly different from those demonstrated by TTC perfusion. TTC immersion staining can, thus, only be used as a reliable marker of cerebral ischaemia damage with post-occlusion survival periods of 24 h. TTC perfusion staining gives results not significantly different from histopathology at 4 h or more post-occlusion but at earlier intervals than 24 h it differs significantly from TTC immersion staining.(ABSTRACT TRUNCATED AT 250 WORDS)

The ultrastructural effects of prolonged normothermic and cold ischemia on the cerebral cortex of the adult rat were investigated. Complete cerebral ischemia was produced by cardiac arrest and the animals' temperature was maintained at 37°C for periods ranging from 0 to 81 hours before electron microscopy preparation. Electron micrographs of cold cerebral ischemia were generated after stabilizing the rat's temperature at 0°C after cardiac arrest for periods ranging from 0 hours to 6 months. A qualitative examination of the electron micrographs shows structural signatures of energy depletion such as vessel leaking and chromatin clumping after 1 hour at 37°C and after 24 hours at 0°C, followed by synapse degradation after 6 hours at 37°C and 1 week at 0°C. Evidence of advanced necrosis was observed after 36 hours at 37°C and 2 months at 0°C. A deep learning algorithm was introduced that demonstrates the temperature dependence of ischemia-induced ultrastructural changes and that can also successfully distinguish between early ischemic changes and advanced necrosis.