Nitric oxide is involved in ischemia-induced apoptosis in brain: A study in neuronal nitric oxide synthase null mice

Nitric oxide can promote or inhibit apoptosis depending on the cell type an d coexisting metabolic or experimental conditions. We examined the impact o f 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 ge nerate nitric oxide from neuronal nitric oxide synthase. Adjacent coronal s ections passing through the anterior commissure were stained with hematoxyl in and eosin or terminal deoxynucleotidyl transferase-mediated dUTP nick en d labeling (TUNEL). Immunoblotting was used to identify changes in the anti - and proapoptotic proteins Bcl-2 and Bax, respectively. Activation of casp ases was assessed by appearance of actin cleavage products using a novel an tiserum directed against 32-kDa actin fragment (fractin). In the neuronal n itric oxide synthase mutant mouse, infarct size and TUNEL positive apoptoti c neurons were reduced compared to the wild-type controls. At 6 h, Bcl-2 le vels in the ischemic hemisphere were increased in mutants but decreased in the wild-type strain. Bax levels did not change significantly. Caspase-medi ated 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-t ype. The reduction in the number of TUNEL and fractin positive apoptotic ce lls appears far greater than anticipated based on the smaller lesion size i n mutant mice. Hence, from these data we suggest that a deficiency in neuronal nitric oxid e production slows the development of apoptotic cell death after ischemic i njury and is associated with preserved Bcl-2 levels and delayed activation of effector caspases. (C) 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.