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

This is the first report on increased neuronal levels of biliverdin reducta se (BVR) in response to ischemic brain injury. BVR is an oxidoreductase, an d 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 ca talyses the final step in the heme metabolic pathway and reduces the heme d egradation product, biliverdin, to bilirubin. Bilirubin can be both a neuro toxicant and an antioxidant depending on its ratio to protein and concentra tion. Bilirubin also has immunomodulatory activity. Other biologically acti ve heme degradation products are iron and CO. This study assessed time-depe ndent changes in the level of BVR, following permanent middle cerebral arte ry occlusion (MCAo). It also examined correlation of the change in BVR expr ession with display of indices of ischemic tissue injury. Under halothane a nesthesia and normothermic conditions, 72 DNX inbred mice were subjected to MCAo. A time-dependent enlargement of an ischemic lesion over the course o f 24 h was observed and measured 55 +/- 5 mm(3) at 6 h, 63 +/- 6.7 mm3 at 1 2 h, and 73 +/- 5 mm3 at 24 h. Six hours after MCAo, increased immunoreacti vity 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 n igra, in the Purkinje layer of the cerebellum and in the central nucleus of inferior colliculus. Twenty-four hours after MCAo, immunoreactivity for BV R remained increased in the peri-ischemia areas. At all time points stainin g 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 increa se 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 hemisph ere, when compared with the contralateral hemisphere, neither measurable de creases in BVR mRNA or total protein levels nor a decrease in NADH-dependen t BVR activity at pH 6.7 were observed. As judged by Northern and Western b lots and activity analysis, despite the apparent loss of BVR from the ische mic core, and its increase in the peri-ischemic region, when compared with the contralateral hemisphere, the overall capacity of the ischemic hemisphe re to catalyze the reduction of biliverdin was unchanged throughout the exp eriment. Should, in the case of ischemia, the conditions favor the antioxid ant 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 ti ghtly regulated expression of BVR in the ischemic hemisphere as an importan t factor in protection against bilirubin neurotoxicity. Data suggest that p harmacological modulation of BVR expression is a possible new direction for protecting neurons against ischemic injury and oxidative stress. (C) 1999 Elsevier Science B.V. All rights reserved.