Genome-Wide Analysis of Müller Glial Differentiation Reveals a Requirement for Notch Signaling in Postmitotic Cells to Maintain the Glial Fate

Previous studies have shown that Müller glia are closely related to retinal progenitors; these two cell types express many of the same genes and after damage to the retina, Müller glia can serve as a source for new neurons, particularly in non-mammalian vertebrates. We investigated the period of postnatal retinal development when progenitors are differentiating into Müller glia to better understand this transition. FACS purified retinal progenitors and Müller glia from various ages of Hes5-GFP mice were analyzed by Affymetrix cDNA microarrays. We found that genes known to be enriched/expressed by Müller glia steadily increase over the first three postnatal weeks, while genes associated with the mitotic cell cycle are rapidly downregulated from P0 to P7. Interestingly, progenitor genes not directly associated with the mitotic cell cycle, like the proneural genes Ascl1 and Neurog2, decline more slowly over the first 10–14 days of postnatal development, and there is a peak in Notch signaling several days after the presumptive Müller glia have been generated. To confirm that Notch signaling continues in the postmitotic Müller glia, we performed in situ hybridization, immunolocalization for the active form of Notch, and immunofluorescence for BrdU. Using genetic and pharmacological approaches, we found that sustained Notch signaling in the postmitotic Müller glia is necessary for their maturation and the stabilization of the glial identity for almost a week after the cells have exited the mitotic cell cycle

Decreased intraocular pressure induced by nitric oxide donors is correlated to nitrite production in the rabbit eye.

PURPOSE: To evaluate the effect of intraocular administration of nitric oxide (NO) donors in the rabbit eye on intraocular pressure (IOP), inflammation, and toxicity. METHODS: Intravitreal and intracameral injections of two NO donors, SIN-1 and SNAP, and SIN-1C and BSS were performed. Clinical examination, IOP measurements, protein evaluation in aqueous humor, and histologic analysis of the ocular globes were realized. Nitric oxide release was demonstrated by nitrite production in the aqueous humor and in the vitreous using the Griess reaction. RESULTS: The drastic decrease of IOP, observed after a single NO donor injection, was correlated directly with nitrite production and, thus, to NO release. Injection of inactive metabolite of SIN-1, SIN-1C, which is not able to release NO, did not modulate IOP. When administered in the aqueous humor or in the vitreous, NO did not diffuse from one segment of the eye to another. No inflammation or histologic damage was observed as a result of a single NO donor administration. CONCLUSIONS: Nitric oxide is implicated directly in the regulation of IOP and its acute, and massive release into the rabbit eye did not induce inflammation or other growth toxic effects on the ocular tissues

Expression of inducible nitric oxide synthase in bovine corneal endothelial cells and keratocytes in vitro after lipopolysaccharide and cytokines stimulation.

PURPOSE: To determine whether bovine corneal endothelial (BCE) cells and keratocytes express the inducible form of nitric oxide synthase (NOS) after exposure to cytokines and lipopolysaccharide (LPS), and to study the regulation of NOS by growth factors. METHODS: Cultures of bovine corneal endothelial cells and keratocytes were exposed to increasing concentrations of LPS, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). At selected intervals after exposure, nitrite levels in the supernatants were evaluated by the Griess reaction. Total RNA was extracted from the cell cultures, and messenger RNA levels for inducible NOS (NOS-2) were measured by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Exposure of BCE cells and keratocytes to LPS and IFN-gamma resulted in an increase of nitrite levels that was potentiate by the addition of TNF-alpha. Analysis by RT-PCR demonstrated that nitrite release was correlated to the expression of NOS-2 messenger RNA in BCE cells and keratocytes. Stereoselective inhibitors of NOS and cycloheximide inhibited LPS-IFN-gamma-induced nitrite release in both cells, whereas transforming growth factor-beta (TGF-beta) slightly potentiated it. Fibroblast growth factor-2 (FGF-2) inhibited LPS-IFN-gamma-induced nitrite release and NOS-2 messenger RNA accumulation in keratocytes but not in BCE cells. CONCLUSIONS: The results demonstrate that in vitro activation of keratocytes and BCE cells by LPS and cytokines induces NOS-2 expression and release of large amounts of NO. The high amounts of NO could be involved in inflammatory corneal diseases in vivo