Freshly sampled brain tissue exposed to
2,3,5-triphenyltetrazolium chloride (TTC) acquires a red color because mitochondrial
enzymes reduce the colorless TTC to a red, water-insoluble
formazan deposit. Pan-necrotic areas remain uncolored, which enables quantitation of experimental
brain injury by optical scanning and image analysis of serial slices to determine the relative volume of red versus infarcted, non-stained, tissue. The accuracy of this method can be challenged, however, when
infarction is accompanied by areas of partial, scattered injury where differences in coloration are difficult to see or quantify. We tested the feasibility of measuring scattered injury using a principle which underlies standard assays for in vitro cell survival, namely extracting deposited
formazan with a
solvent and measuring its level by spectrophotometry. Anesthetized, adult Sprague Dawley rats were subjected to 12 min of
cerebral ischemia to produce selective, delayed neuronal death in hippocampus, striatum and cortex. Some rats also received 6 h of whole-body
hypothermia treatment (31.5-32.5 degrees C) immediately after
ischemia.
Ischemia rats and non-operated controls were sacrificed 1 week later. Hippocampus and portions of cerebrum were incubated 90 min in a 2% TTC
solution and then soaked in a measured volume of 50:50
ethanol and
dimethylsulfoxide to extract the red
formazan product. Spectrophotometric measurements of the extract showed a diminished
formazan coloration (absorbance/g brain) in all samples from the untreated
ischemia group compared to non-operated controls. This apparent
brain injury was attenuated in the group of
ischemia rats that received
hypothermia treatment. We conclude that
solvent extraction and spectrophotometric quantitation of
formazan has potential utility as an objective way to index experimental
brain injury even if this is diffuse in nature and not amenable to measurement by conventional image analysis techniques.