title: 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.
authors.affiliation: Departments of Ophthalmology, College of Medicine, University of Florida, Gainesville 32610-0284, USA.
authors.forename: X
authors.initials: X
authors.lastname: Qi
authors.forename: J
authors.initials: J
authors.lastname: Guy
mesh_terms: D000818:Animals; D005260:Female; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis*; D006168:Guinea Pigs / Q000378:metabolism*; D006651:Histocytochemistry / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D008853:Microscopy / Q000379:methods; D008854:Microscopy, Electron; D009252:NADPH Dehydrogenase / Q000032:analysis*; D019001:Nitric Oxide Synthase / Q000032:analysis*; D009900:Optic Nerve / Q000201:enzymology*; D012016:Reference Values
chemical_list: D005904:Glial Fibrillary Acidic Protein; D019001:Nitric Oxide Synthase; D009252:NADPH Dehydrogenase