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E. Aguilar, V. Marchetti, M. S. Wang, A. L. Dorsey, T. U. Krohne, M. Friedlander; In vivo and Ex Vivo Imaging of Endogenous Retinal Myeloid Cells in Retinal Development and Oxygen-Induced Retinopathy in the CX3CR1+/eGFP Mouse. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5767.
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© ARVO (1962-2015); The Authors (2016-present)
Endogenous retinal myeloid cells perform crucial functions in retinal angiogenesis. We studied the role of these cells in vascular developmental and pathological vascular remodeling using both in vivo and ex vivo imaging techniques.
We used heterozygous transgenic mice (CX3CR1+/eGFP) in which one allele of the CX3CR1 gene is replaced by the gene encoding enhanced green fluorescent protein (eGFP), thus resulting in eGFP expression in all cell of the myeloid lineage. Retinas of these animals were analyzed both in vivo using a confocal scanning laser ophthalmoscopy (cSLO; Spectralis, Heidelberg Engineering) and ex vivo by means of immunohistochemistry and confocal microscopy. For retinal developmental studies, animals were examined at different ages from postnatal day 0 (P0) to P21. For the oxygen-induced retinopathy (OIR) model, animals were subjected to 75% oxygen at P7-P12 and subsequently analyzed at P12, P14, and P17. In additional experiments, animals received an intravitreal injection of human cord blood CD14+ cells at P7 prior to OIR treatment.
eGFP+ endogenous cells were present on the retinal surface at P0 and in the intermediate and deep plexus starting at P5-P7. eGFP+ cells were positive for isolectin Griffonia simplicifolia. The number and distribution of these cells became more widespread with increasing animal age. In the OIR model, retinas exhibited a decreased density of eGFP+ cells in the avascular areas and decreased cellular binding of isolectin by these cells, suggesting diminished cell activation. High oxygen exposure decreased the level of association of eGFP+ cells with the vasculature and reduced their number both in the superficial and in deep plexus. Injection of CD14+ cells re-established the number, activation level, and distribution pattern of eGFP+ cells in all layers of the retina.
In vivo imaging techniques using cSLO allow for high-resolution analysis of cellular morphologies and distribution patterns in the rodent retina that correlate well with immunohistochemical observations and, in addition, permit longitudinal studies in live animals.
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