Rapid quantification of ischaemic injury and cerebroprotection in brain slices using densitometric assessment of 2,3,5-triphenyltetrazolium chloride staining

Abstract

2,3,5-Triphenyltetrazolium chloride (TTC), a marker of mitochondrial enzyme activity, is widely used to assess the effects of cerebral ischaemia in vivo. In the present study, we characterised its utility as a simple rapid macrohistological measure of ischaemic damage in brain slices. Coronal rat corticostriatal slices were incubated in oxygenated artificial cerebrospinal fluid (aCSF) until subjected to 'ischaemia' (deoxygenated, hypoglycaemic aCSF) for up to 12 min. After a further 30 min to 16 h of reincubation in oxygenated aCSF, slices were stained with TTC, fixed with formalin and transferred to cover slips. The slices were scanned in 8-bit greyscale using a standard desktop scanner and the staining analysed by densitometry of the acquired images. Control slices stained a rich pink/red. Ischaemia (10 min) reduced both the area and intensity of staining. Both measures of striatal staining were negatively correlated with the duration of ischaemia (0-12 min). Furthermore, staining in the striatum correlated significantly with cortical TTC staining. The effects of TTC concentration (0.063-0.5% w/v) and post-ischaemic interval (30 min to 16 h) were examined upon the intensity of TTC staining. (+)-MK 801 prevented the ischaemia-induced reduction in TTC staining, consistent with cerebroprotection. These data suggest that TTC staining of brain slices may be used to quantify ischaemic injury and cerebroprotection.