Immunolocalization of nitric oxide synthase in the retinas of five mammalian species

Purpose: This study was undertaken to determine the the immunostaining patterns for three isoforms of Nitric Oxide Synthase (NOS) in normal retinal tissues from five mammalian species. Methods: A pannel of synthetic peptide generated, isoform specific, antibody probes were used to immunolocalize b-NOS (NOS 1), iNOS (NOS 2), and ccNOS (NOS 3) in fixed tissues from normal retinas. Retinal tissues, including the RPE and choroid, from human, cat, dog, rabbit and rat were fixed in 4% paraformaldehyde, and embedded in either agarose or paraffin. Light microscopic immunocytochemistry was performed on tissue cross sections by a robotic immunostainer (TechMate 1000, BioTek Solutions). A laser scanning confocal microscope was used to examine agarose embedded, sections. Paraffin sections were examined with a Zeiss PMIII phoromicroscope. Results: Staining was highly similar across all species examined when antibodies to the neuronal (NOS 1) or endothelial cell (NOS 3) forms of the enzyme were used. Areas of positive immunolabeling were observed in the ganglion cells, the inner plexiform and inner nuclear layers, the outer plexiform layer, the photoreceptor inner segments and the vessels of the choroid. In certain species, some positive staining was also evident in the Müller's glia and the RPE. No tissue labeling was observed in any species when an antibody specific to the induced (NOS 2) isoform of the enzyme was used. Conclusions: Nitric oxide is a multifunctional molecule which can interact with various tissue types. The presence of the two constituative isoforms of NOS in normal ocular tissues, especially at or near sights of synaptic transmission, strongly suggests that NO may act to modulate neuronal activity in normal retina.