TTC, Fluoro-Jade B and NeuN staining confirm evolving phases of infarction induced by middle cerebral artery occlusion

Considerable debate exists in the literature on how best to measure infarct damage and at what point after middle cerebral artery occlusion (MCAO) infarct is histologically complete. As many researchers are focusing on more chronic endpoints in neuroprotection studies it is important to evaluate histological damage at later time points to ensure that standard methods of tissue injury measurement are accurate. To compare tissue viability at both acute and sub-acute time points, we used 2,3,5-triphenyltetrazolium chloride (TTC), Fluoro-Jade B, and NeuN staining to examine the evolving phases of infarction induced by a 90-min MCAO in mice. Stroke outcomes were examined at 1.5h, 6h, 12h, 24h, 3d, and 7d after MCAO. There was a time-dependent increase in infarct volume from 1.5h to 24h in the cortex, followed by a plateau from 24h to 7d after stroke. Striatal infarcts were complete by 12h. Fluoro-Jade B staining peaked at 24h and was minimal by 7d. Our results indicated that histological damage as measured by TTC and Fluoro-Jade B reaches its peak by 24h after stroke in a reperfusion model of MCAO in mice. TTC staining can be accurately performed as late as 7d after stroke. Neurological deficits do not correlate with the structural lesion but rather transient impairment of function. As the infarct is complete by 24h and even earlier in the striatum, even the most efficacious neuroprotective therapies are unlikely to show any efficacy if given after this point.