Spectrophotometric measurement of experimental brain injury

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.