Expression of the inducible form of nitric oxide synthase by reactive astrocytes after transient global ischemia

We recently demonstrated that reactive astrocytes express NADPH diaphorase activity, a marker for Nitric Oxide Synthase, following transient global ischemia (Neuroscience Letters 154: 125-128). There has been little evidence that astrocytes express Nitric Oxide Synthase or produce NO (nitric oxide) in vivo; although in vitro experiments have shown that cultured astrocytes can produce NO. To determine whether reactive astrocytes express inducible form of NOS (iNOS) in vivo, we studied the pathological changes of rat hippocampus by immunohistochemistry after 10 minutes of transient global ischemia, which results in the selective delayed death of CA1 pyramidal cells and marked gliosis in the CA1 subfield. In the normal hippocampus, astrocytes express neither NADPH diaphorase activity nor iNOS. After ischemia, the temporal and spatial pattern of iNOS, NADPH diaphorase, and GFAP are very similar, indicating that reactive astrocytes express iNOS. Double staining for NADPH diaphorase and GFAP, or iNOS and GFAP confirmed that reactive astrocytes express both NADPH diaphorase activity and iNOS immunoreactivity. These changes were observed three day after ischemia and increased in prominence from one week to one month. The staining pattern of OX42, an antibody that recognizes both microglia and macrophages, is spatially and temporally distinct from the pattern of NADPH diaphorase and iNOS staining. Thus, we conclude that transient global ischemia induces iNOS primarily in reactive astrocytes. This increase in NOS expression and, presumably, NO production by reactive astrocytes may play a role in the process of delayed neuronal death or in the remodeling responses that occur after ischemic damage.