Use of TTC staining for the evaluation of tissue injury in the early phases of reperfusion after focal cerebral ischemia in rats

Background and purpose: 2,3,5-Triphenyltetrazolium chloride (TTC) staining measures tissue viability used to evaluate infarct size. The goal of this study was to compare viability of neuronal tissue during the early phases of ischemia–reperfusion assessed either by perfusion of the brain with TTC solution transcardially, in vivo, or by staining brain slices, in vitro. Methods: The middle cerebral artery was occluded for 1 h in male SPRD rats and then reperfused for 0, 1, 4, 8, 16 and 24 h. Ischemic damage was evaluated by TTC staining, in vivo and in vitro, and by histology (Luxol Fast Blue and Fluoro-Jade staining, electron microscopy). Results: Core volume of tissue injury measured in vivo was large at 0 h and steadily decreased by 50% ( p < 0.001) up to 16 h, but substantially increased from 16 to 24 h of reperfusion. In contrast, a significant core volume appeared at 4 h only, in vitro, and gradually increased up to 24 h. Core volume was larger in vivo than in vitro at all times except at 16 h when the opposite was observed. Evans blue administered intracardially stained TTC-negative areas at 1 and 24 h. Histology covered the evolution of serious tissue injury but also demonstrated some morphologically preserved neurons in the infracted area at 24 h. Conclusions: Formation of formazan from TTC can depend on both the staining method and the metabolic burden of the brain tissue causing uncertainties in the volume of ischemia-induced brain injury measured by TTC staining.