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I. A. Bhutto, T. Baba, M. Edwards, C. Merges, D. S. McLeod, G. A. Lutty; Immunohistochemical Localization of Neuronal and Endothelial Nitric Oxide Synthase in Fetal Human Eye. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5949.
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Nitric oxide (NO) is a potent vasodilator and important modulator of cellular functions in both neuronal and non-neuronal cells. Vasodilation is an important step in the formation and growth of new blood vessels. The purpose of this study was to investigate the expression of neuronal (nNOS) and endothelial (eNOS) nitric oxide synthase during development of human embryonic and fetal eyes.
Alkaline phosphatase immunohistochemistry was performed on cryopreserved sections of human eyes from 7 to 21 weeks gestation (WG) using antibodies against nNOS and eNOS. CD31 antibody was used as an endothelial cell (EC) marker and CXCR4 as a marker of retinal progenitors and angioblasts. Immunofluorescence and confocal microscopy was used for double labeling to further confirm the nNOS nuclear localization in vascular and nonvascular cells.
At 7 WG, nNOS immunoreactivity was weak and scattered in nuclei of CXCR4+ progenitors within the inner neuroblastic layer of the retina and in mesenchymal precursors in choroid, as well as in EC nuclei of the tunica vasculosa lentis (TVL). At 12 WG, nNOS reactivity was prominent in nuclei of endothelial cells of TVL, migrating angioblasts in nerve fiber layer, progenitors in neuroblastic layer and in RPE nuclei. In choroid immunoreactivity was observed in mesenchymal cells and choriocapillaris (CC). By 21 WG, nNOS immunostaining was mostly associated with cells within the ganglion cell layer, as well as ECs of developing retinal blood vessels and RPE nuclei. eNOS immunoreactivity was confined to ECs of retinal and choroidal developing vessels and TVL at all ages studied. By confocal microscopy, we confirmed the nNOS nuclear localization with DAPI and angioblast association by colocalization with CXCR4.
In fetal human eyes, nNOS was prominently expressed in nuclei of RPE and vascular ECs in retina and choroid and TVL. These findings suggest that nuclear nNOS may have a role in the transcription regulatory system in undifferentiated angioblasts and differentiated endothelial cells during fetal development. Interestingly, RPE have nuclear nNOS immunolabeling at all ages studied, even the adult (Bhutto et al. Exp Eye Res. 2010;90:155-167). Detailed studies on the role of nuclear nNOS are necessary to further elucidate NO/nNOS pathways.
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