NADPH-diaphorase activity in activated astrocytes represents inducible nitric oxide synthase

In paraformaldehyde-fixed sections of healthy brain, glial cells at the light-microscope level do not contain measurable levels of NADPH-diaphorase. However, after a variety of lesions in the mouse brain, some reactive astrocytes express varying amounts of this enzyme. Following stab wounds, activated astrocytes or related glial cells surrounding the lesion, contained moderate to high levels of NADPH-diaphorase in the cerebellum, midbrain, thalamus, striatum, hippocampal formation and neocortex. Double-labelling experiments confirmed that this corresponds to an inducible form of nitric oxide synthase, similar to that found in activated macrophages. Within the lesion there were large numbers of macrophages which also contained NADPH-diaphorase. After 10 min of global hypoxic ischaemia, some reactive astrocytes also contained NADPH-diaphorase. These cells were confined to the dorsal part of the hippocampal formation (the dentate fascia and CA1 areas) and to the anterolateral striatum. More focal ischaemic damage, produced by dividing an arterial branch, also produced a rim of reactive astrocytes containing NADPH-diaphorase, that surrounded the area of necrosis. Low levels of NADPH-diaphorase were induced within one day of a stab wound and the enzyme activity reached near maximal levels by two days postlesion. Moderate NADPH-diaphorase activity was still present at 63 days postlesion, but only a small number of astrocytes were stained in the immediate vicinity of the lesion. These experiments confirm that NADPH-diaphorase activity represents inducible nitric oxide synthase in activated astrocytes and probably in inflammatory macrophages. We conclude that a high proportion of activated astrocytes and a small proportion of invading macrophages are induced to express moderate to high levels of nitric oxide synthase following neuronal damage. Our results indicate that following a variety of lesions reactive astrocytes are synthesizing significant levels of nitric oxide within 24 h. This nitric oxide may be involved in modulating the likelihood of epileptic seizures.