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T. J. Giove, C. S. Marbach, W. D. Eldred; Alterations in Neuronal and Inducible Nitric Oxide Synthase in the Diabetic Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4968.
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This study examines how the expression levels of neuronal and inducible nitric oxide synthase (nNOS and iNOS) and VEGF change in the progression of early diabetic retinopathy.
Diabetes was induced in adult male mice by streptozotocin injection. Mice were then sacrificed at 3, 5, and 20 weeks after onset of diabetes and their eyes were surgically removed. The resultant eyecups were fixed, embedded, cryosectioned, and subsequently used for immunocytochemistry or NADPH-diaphorase histochemistry. In addition, protein was extracted from some retinas for western blots.
Immunocytochemistry shows a dramatic change in the subcellular localization of nNOS in the inner plexiform layer (IPL) at all time points in the diabetic retina. In control retinas, the nNOS is found to fill many dendritic arborizations and in many apparent synaptic boutons. In diabetic retinas, the nNOS is almost exclusively localized within synaptic boutons. In contrast, there are no large differences in nNOS protein levels seen in western blots. NADPH-diaphorase staining shows a marked increase in the diabetic retinas, and the staining pattern closely mirrors the nNOS immunoreactivity. Levels of iNOS and VEGF are seen to increase at 5 weeks.
Both nNOS and iNOS appear to be modulated in the diabetic mouse retina within 3-5 weeks after diabetic onset. nNOS appears to concentrate heavily in the synaptic boutons in the IPL in diabetic animals, with almost none remaining in the dendritic processes. The western blots suggest that there is not less nNOS, but a change in either the translocation or the location of synthesis for the protein. The elevated NADPH-diaphorase levels and staining pattern supports the idea that nNOS is more active in the diabetic retina. The increase in iNOS by 5 weeks coincides with the elevation of VEGF. Taken together, this data suggests that changes in nNOS and iNOS begin early in diabetic retinopathy and may be independent of vascular complications in the case of nNOS.
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