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A.A. M. Assiri, M.A. Wride, F.C. Mansergh, V.E. Walker, R.D. Young, M.J. Evans, A.J. Quantock; Corneal stromal ultrastructure in mice with Sparc–null mutations. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3822.
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Purpose:Sparc (secreted protein acidic and rich in cysteine, also known as osteonectin/BM40) is a glycoprotein that interacts with a range of extracellular matrix molecules. Mice deficient for Sparc show severe age–onset cataract formation and disruption of the lens (Gilmour et al., EMBO J 1998;17:1860–1870). They also display abnormalities in the dermis (Bradshaw et al., J Invest Dermatol 2003;120:949–955). The current study was designed to investigate the internal fine structure of the corneal stroma in targeted mice produced in our laboratory that are homozygous null for Sparc. Methods: Corneas from wild–type (129Sv/Ev) mice and from Sparc–null mutants bred on pure (Sp–/– 129Sv/Ev) or mixed outbred (Sp–/–129Sv/Ev/MF1bb) backgrounds were fixed and embedded for light microscopy and transmission electron microscopy. Cupromeronic blue at a critical electrolyte concentration of 0.1M MgCl2 was sometimes included in the fixative as a cationic stain for sulphated proteoglycans. Ultrathin sections were stained with 0.5% aqueous uranyl acetate followed by 1% phosphotungstic acid. Results: Corneas of Sparc–null mutants appeared significantly thinner than normal, with evidence in histologic sections of distended corneas. Collagen fibril diameters were unchanged, measuring 28.2nm (n=1049, SD=3.6nm) in wild types and 28.2nm (n=1138, SD=4.3nm) in Sp–/– 129Sv/Ev mutants. Proteoglycan staining in mutants, on occasion, looked to be affected with less filaments in evidence. The most striking finding, however, was of heightened collagen fibril spacing in Sparc null corneas. This was more noticeable in mutants bred on a pure rather than a mixed outbred background, and was much more extensive in deeper tissue regions. Conclusions: Other investigators have shown that an absence of Sparc results in abnormally thin collagen fibrils in skin (Bradshaw et al., J Invest Dermatol 2003;120:949–955). Our finding of normal diameter collagen fibrils in the corneal stroma of Sparc null mutants is suggestive of different regulatory mechanisms for collagen fibrillogenesis in these tissues. Sparc is known to accumulate during corneal repair (Berryhill et al. Exp Eye Res 2003;77:85–92). The current data indicate that the absence of Sparc adversely affects the ultrastructural arrangement of collagen fibrils in the corneal stroma.
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