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T. Baba, D. S. McLeod, C. Merges, I. A. Bhutto, R. Grebe, M. Edwards, E. F. Wawrousek, G. A. Lutty; -Crystallin Knockout Mice Have Normal Vascular Development in Advance of a Delayed Astrocyte Template. Invest. Ophthalmol. Vis. Sci. 2010;51(13):33.
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© ARVO (1962-2015); The Authors (2016-present)
During primary retinal vascular development in rodents, an astrocyte template has been reported to be critical for guiding endothelial tip cells. Recently, a small heat shock protein, αB-crystallin (CryαB) was reported to regulate glial fibrillary acidic protein (GFAP) assembly in astrocytes [Hageman TL et al. Hum Mol Genet 2009; 18: 1190-9]. We used CryαA/CryαB/heat shock protein-b2 knockout mice (α-cry -/-) to investigate the role of α-cry’s in retinal astrocyte development, and the relationship between astrocytes and primary blood vessels during early stages of retinal vasculogenesis.
α-cry -/- mice (n= 8, from P3 to P35) and wild-type (WT) controls (129S6/SvEvTac, n= 4) were used under the ARVO statement for the use of animals in ophthalmic and vision research. Flatmount retinas labeled with Isolectin B4, as a vascular marker, and GFAP, as an astrocyte marker, were examined by confocal microscopy. Morphometric analysis was performed to measure the distance from optic nerve head to the peripheral edge of Isolectin-labeled vasculature and GFAP-positive astrocytes and the number of vascular branches and filopodia of endothelial tip cells.
The GFAP-labeled astrocytes were observed in advance of endothelial cells in 98% of the measurements in WT retinas and 56% of those in α-cry -/- retina. There was a significant difference in the distance from the optic nerve head to vascular or astrocyte front in α-cry -/- mice compared to WT (P< 0.0001). In 44% of measurements in α-cry -/- retina, blood vessels were 74.8 +/- 45.1 µm in advance of GFAP-labeled astrocytes. The number of vascular branches and endothelial cell filopodia were not different between WT and α-cry -/- (P= 0.358, P= 0.683). In α-cry -/- retina, the vascular network had reached the ora serrata by around P7 and the primary vascular pattern was normal at P7, P14 and P35.
There was a delay in formation of a GFAP-positive astrocyte template in α-cry -/- mice. However, the development of peripheral vasculature was similar to WT, suggesting there are processes other than an astrocyte template which guide vasculogenesis in developing retina.
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