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Nitric oxide is involved in ischemia-induced apoptosis in brain: a study in neuronal nitric oxide synthase null mice
Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.
Nitric oxide can promote or inhibit apoptosis depending on the cell type and coexisting metabolic or experimental conditions. We examined the impact of nitric oxide on development of apoptosis 6, 24, and 72 h after permanent middle cerebral artery occlusion in mutant mice that lack the ability to generate nitric oxide from neuronal nitric oxide synthase. Adjacent coronal sections passing through the anterior commissure were stained with hematoxylin and eosin or terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Immunoblotting was used to identify changes in the anti- and proapoptotic proteins Bcl-2 and Bax, respectively. Activation of caspases was assessed by appearance of actin cleavage products using a novel antiserum directed against 32-kDa actin fragment (fractin). In the neuronal nitric oxide synthase mutant mouse, infarct size and TUNEL positive apoptotic neurons were reduced compared to the wild-type controls. At 6 h, Bcl-2 levels in the ischemic hemisphere were increased in mutants but decreased in the wild-type strain. Bax levels did not change significantly. Caspase-mediated actin cleavage appeared in the ischemic hemisphere at this time point, and was significantly less in mutant brains at 72 h compared to the wild-type. The reduction in the number of TUNEL and fractin positive apoptotic cells appears far greater than anticipated based on the smaller lesion size in mutant mice.Hence, from these data we suggest that a deficiency in neuronal nitric oxide production slows the development of apoptotic cell death after ischemic injury and is associated with preserved Bcl-2 levels and delayed activation of effector caspases.
Actins/metabolism Animals Apoptosis/*physiology Brain/*enzymology/pathology/physiopathology Brain Ischemia/*enzymology/pathology/physiopathology Caspases/metabolism Coloring Agents/pharmacology DNA Fragmentation/physiology Eosine Yellowish-(YS)/pharmacokinetics Female Hematoxylin/pharmacokinetics Immunohistochemistry In Situ Nick-End Labeling Male Mice Mice, Knockout Neurons/*enzymology/pathology Nitric Oxide/*physiology Nitric Oxide Synthase/*metabolism Proto-Oncogene Proteins/metabolism Proto-Oncogene Proteins c-bcl-2/metabolism bcl-2-Associated X Protein
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