Correlation of triphenyltetrazolium chloride perfusion staining with conventional neurohistology in the detection of early brain ischaemia.

Triphenyltetrazolium chloride (TTC) has been used to detect experimental brain ischemia but its accuracy has not been established fully, especially in models of early brain ischaemia. We have developed a technique that combines TTC-staining and perfusion-fixation with formalin in the same specimen. The left middle cerebral artery of 12 rats, anaesthetized with halothane were exposed via a subtemporal approach and occluded for 4 h. The animals were perfused transcardially with TTC (2%) and formalin (10%). The forebrain was sliced and the size of the ischaemic area, delineated by TTC, was measured. The brain slices were then processed for light microscopy, and the amount of ischaemic damage determined. The ischaemic volumes of the hemisphere and cortex assessed by TTC (127.4 +/- 21.7 mm3 and 18.4 +/- 3.0 mm3) were larger than the volumes measured by microscopy (104.3 +/- 16.6 mm3 and 15.2 +/- 3.6 mm3) but there was no statistical difference between them. Correlation of the ischaemic volumes in hemisphere and cortex between the two methods was good (r2 = 0.9221, P less than 0.01 and r2 = 0.9243, P less than 0.001), but the correlation of the ischaemic areas at specific coronal planes was poor. The ischaemic volume of caudate nucleus measured by TTC (15.2 +/- 3.6 mm3) was smaller than the volume assessed by microscopy (18.4 +/- 3.0 mm3) and the correlation was poor (r2 = 0.6650) It is concluded that TTC-staining provides only an approximation of the amount of early ischaemic brain damage subsequently identified by conventional light microscopy.