Comparative study of 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin-eosin staining for quantification of early brain ischemic injury in cats

There are no staining methods that can reliably and unequivocally detect final infarcts in the acute stage of experimental ischemia. In most instances, 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin and eosin (HE) stainings are accepted for this purpose, but neither is perfect. We performed a comparative study of the TTC immersion method and HE staining for quantification of early brain ischemic injury in cats, focussing on the reproducibility associated with planimetry. Focal brain ischemia was produced by middle cerebral artery (MCA) occlusion via the transorbital approach in 14 cats. After 6 h of occlusion, two slices of 3 mm thickness, passing through the optic chiasma and mammillary body, were selected for pathological examination. TTC immersion and HE staining were both used for planimetric study of the surface of the same slice. The area of the injury was traced manually with the aid of computerized digital planimetry and expressed as a percentage of the area of the contralateral hemisphere. The area of hemispheric injury and the area of gray matter injury were separately calculated in the TTC specimens, and each was compared with the area of gray matter injury in the HE-stained specimens. Measurements were repeated twice on each slice to estimate errors associated with manual tracing of the boundary of the area of injury. The mean percentage values of the area of injury in the TTC-immersed gray matter specimens were lower than those detected by HE staining, although there was a very significant correlation between the two. The differences between each two measurements of TTC-determined gray matter injury were significantly less than those between each two measurements of HE-determined gray matter injury. The differences between each two measurements of TTC-determined hemispheric injury were slightly less than those between each two measurements of TTC-determined gray matter injury, although there was no significant difference between them. For quantifying ischemic injury after 6 h of MCA occlusion in cats, the TTC immersion method is more reproducible and simpler in manner than HE staining, but the results of both are significantly correlated.