Triphenyltetrazolium chloride (TTC) as a marker for ischaemic changes in rat brain following permanent middle cerebral artery occlusion

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Abstract
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. Thus at times of less than 24 h it may reflect mainly changes in stain perfusion, rather than ischaemic damage.