Detection of hypoxic cells with the 2-nitroimidazole, EF5, correlates withearly redox changes in rat brain after perinatal hypoxia-ischemia

The hypoxia-dependent activation of nitroheterocyclic drugs by cellular nit roreductases leads to the formation of intracellular adducts between the dr ugs and cellular macromolecules. Because this covalent binding is maximal i n the absence of oxygen, detection of bound adducts provides an assay for e stimating the degree of cellular hypoxia in vivo. Using a pentafluorinated derivative of etanidazole called EF5, we studied the distribution of EF5 ad ducts in seven-day-old rats subjected to different treatments which decreas e the level of oxygen in the brain. EF5 solution was administered intraperi toneally 30 min prior to each treatment. The effect of acute and chronic hy poxia on FF5 adduct formation (binding) was studied in the brain of newborn rats exposed to global hypoxia (8% O-2 for 30, 90 or 150 min) and in the b rain of chronically hypoxic rat pups with congenital cardiac defects (Wista r Kyoto). The effect of combined hypoxia-ischemia was investigated in rat p ups subjected to right carotid coagulation and concurrent exposure to 8% O- 2 for 30, 90 or 150 min. Brains were frozen immediately at the end of each treatment. Using a Cy3-co njugated monoclonal mouse antibody (ELK3-51) raised against EF5 adducts, hy poxic cells within brain regions were visualized by fluorescence immunocyto chemistry. Brains from controls or vehicle-injected animals showed no EFS b inding. Notably, brains from animals which were chronically hypoxemic as a result of congenital cardiac defects also showed no EF5 binding. A short ex posure (30 min) to hypoxia or to combined hypoxia-ischemia resulted in incr eased background stain and few scattered cells with low-intensity immunosta ining. Acute hypoxia exposure of at least 90-150 min, which in this age ani mal does not result in frank cellular damage, produced patchy areas of low- to moderate-intensity fluorescence scattered throughout the brain. In cont rast, 90-150 min of hypoxia-ischemia was associated with intense immunofluo rescence in the hemisphere ipsilateral to the carotid occlusion, with a pat tern similar to that reported previously for the histological damage seen i n this model. This study provides a sensitive method for the evaluation of the level of o xygen depletion in brain tissue after neonatal hypoxia-ischemia, at times m uch earlier than any method demonstrates apoptotic or necrotic cell death. Since the level of in vivo formation of macromolecular adducts of EF5 depen ds on the degree of oxygen depletion in a tissue, intracellular EF5 binding may serve as a useful marker of regional cellular vulnerability and redox state after brain injury resulting from hypoxia-ischemia. (C) 1999 IBRO. Pu blished by Elsevier Science Ltd.