Enhanced neuronal expression of the oxidoreductase — biliverdin reductase — after permanent focal cerebral ischemia

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This is the first report on increased neuronal levels of biliverdin reductase (BVR) in response to ischemic brain injury. BVR is an oxidoreductase, and is unique among all enzymes characterized to date in having dual pH/dual cofactor requirements — NADH and NADPH at 6.7 and 8.7, respectively. BVR catalyses the final step in the heme metabolic pathway and reduces the heme degradation product, biliverdin, to bilirubin. Bilirubin can be both a neurotoxicant and an antioxidant depending on its ratio to protein and concentration. Bilirubin also has immunomodulatory activity. Other biologically active heme degradation products are iron and CO. This study assessed time-dependent changes in the level of BVR, following permanent middle cerebral artery occlusion (MCAo). It also examined correlation of the change in BVR expression with display of indices of ischemic tissue injury. Under halothane anesthesia and normothermic conditions, 72 DNX inbred mice were subjected to MCAo. A time-dependent enlargement of an ischemic lesion over the course of 24 h was observed and measured 55±5 mm 3 at 6 h, 63±6.7 mm 3 at 12 h, and 73±5 mm 3 at 24 h. Six hours after MCAo, increased immunoreactivity for BVR was noted in neurons in the peri-ischemic areas, intraischemic cortical layers 3 and 5, as well as in neurons in regions distant from the borders of vascular distribution of the MCA, such as those in substantia nigra, in the Purkinje layer of the cerebellum and in the central nucleus of inferior colliculus. Twenty-four hours after MCAo, immunoreactivity for BVR remained increased in the peri-ischemia areas. At all time points staining for BVR was decreased in the ischemic core. At the 24 h time point there was an increase in Fe staining in the perimeter of the lesion and an increase in Schiff's staining for lipid peroxidation at the rim of the lesion. In situ hybridization analysis demonstrated a time dependent increase in BVR mRNA labeling in neurons of the peri-ischemic area. In the ischemic hemisphere, when compared with the contralateral hemisphere, neither measurable decreases in BVR mRNA or total protein levels nor a decrease in NADH-dependent BVR activity at pH 6.7 were observed. As judged by Northern and Western blots and activity analysis, despite the apparent loss of BVR from the ischemic core, and its increase in the peri-ischemic region, when compared with the contralateral hemisphere, the overall capacity of the ischemic hemisphere to catalyze the reduction of biliverdin was unchanged throughout the experiment. Should, in the case of ischemia, the conditions favor the antioxidant activity of bilirubin, then we suggest that increase in BVR expression in ischemic penumbra may present a cellular defense mechanism against free radical-mediated neuronal damage. Furthermore, we interpret the apparent tightly regulated expression of BVR in the ischemic hemisphere as an important factor in protection against bilirubin neurotoxicity. Data suggest that pharmacological modulation of BVR expression is a possible new direction for protecting neurons against ischemic injury and oxidative stress.