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J. Wang, S. Jiang, X. Li, J. Ge; Activation of Nf-B P65 Protects Retinal Ganglion Cells After Retinal Ischemia-Reperfusion in Rat. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4148.
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To investigate whether NF-ΚB p65 is activated and the activation of NF-ΚB p65 protects retinal ganglion cells (RGCs) after retinal ischemia-reperfusion (I/R) in rat.
Retinal ischemia was induced by transiently elevating the intraocular pressure. Animals were euthanized at different time points after the insult. Eyes were enucleated for semiquantitative real-time RT-PCR studies of NF-ΚB p65, IΚB- (endogenous inhibitor of NF-ΚB). Retinal paraffin sections were prepared for immunohistochemistry for phosphor-NF-ΚB p65, phosphor-IΚB-, and ED1 (a marker of microglial/phagocytic cells) antibodies. Two inhibitors of NF-ΚB, helenalin and carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132), and small interfering RNA (siRNA) of NF-ΚB p65 were given to animals to examine the expression of NF-ΚB p65 mRNA and the number of RGCs by cresyl violet staining and 3% Fluoro-Gold staining. The number of RGCs was counted.
Semiquantitative real-time RT-PCR showed elevated NF-ΚB p65 mRNA (n=6) levels at 2 to 12 hours. IΚB- mRNA (n=6) showed elevated levels at 8 hours, compared with that of normal (n=10). Immunoreactivity (IM) of phosphor-NF-ΚB p65 increased in the retinal ganglion cell layer, the inner plexiform layer and the inner nuclear layer showing at 8 and 12 hours respectively after I/R injury. NF-ΚB p65 and ED1 co-localized in microglia/macrophage-like cells. Phospho-IΚB- co-localized with ED1 in the same cells. Helenalin and MG-132 inhibited the expression of NF-ΚB p65 mRNA and decreased significantly the number of RGCs compared with that of the control (p<0.05). Down-expression of NF-ΚB p65 mRNA was detected using siRNA of NF-ΚB p65, which reduced significantly the number of RGCs compared with that of the vehicle and control (p<0.05).
After retinal ischemia-reperfusion injury, NF-ΚB p65 is activated and the activation of NF-ΚB p65 in microglia/macrophage-like cells protects retinal ganglion cells in rat.
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