Localization of NADPH diaphorase/nitric oxide synthase in the optic nerve of the normal guinea pig: A light and electron microscopic study

Abstract

The aim of this study is to determine the presence and subcellular distribution of NADPH diaphorase (NADPH-d)/nitric oxide synthase (NOS) in the optic nerve of the normal guinea pig. Optic nerve specimens were stained by NADPH-d histochemistry, and double labeled by combining NADPH-d histochemistry with immunostaining for (a) anti-glial fibrillary acidic protein (GFAP) antibody for recognition of astrocytes, (b) griffonia simplicifilia B4-isolectin (GSA-IB4) horse radish peroxidase (HRP)-conjugate for identification of microglia, or (c) oligodendrocyte-associated antibodies to carbonic anhydrase isoenzyme II (CA-II) or to galactocerebroside (GalC) for visualization of oligodendrocytes. In addition, constitutive NOS (cNOS) and inducible NOS (iNOS) immunostaining were used for colocalization with NADPH-d histochemistry. Light microscopy revealed NADPH-d reaction product in the blood vessels and neuroglia of the unmyelinated optic nerve head and myelinated retrobulbar optic nerve. Double labeling with GFAP immunoperoxidase combined with NADPH-d histochemistry revealed both activities in astrocytes. Microglia were labeled with GSA-IB4 isolectin HRP-conjugate, but they did not have NADPH-d activity. Oligodendroglia were immunolabeled with anti CA-II or anti GalC antibodies, but they did not have NADPH-d activity. Both iNOS and cNOS immunoperoxidase labeled astrocytes, but not microglia or oligodendroglia. Under transmission electron microscopy, NADPH-d reaction product appeared as electron-dense particles. These particles were seen in the cytoplasm of endothelial cells, perivascular smooth muscle cells and fibrous astrocytes. Axons and myelin were devoid of NADPH-d activity. This study demonstrates the existence and cellular distribution of NADPH-d/NOS activity in endothelial cells, perivascular smooth muscle cells and fibrous astrocytes of the optic nerve of the normal guinea pig. The presence of these non-neuronal sources of NOS in the optic nerve provides the foundation for future comparative studies of the functional role of reactive oxygen induced toxicity in disorders affecting the optic nerve.