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R. Young, C. Boote, S. K. Swamynathan, A. J. Quantock, J. Piatigorsky, K. M. Meek; Ultrastructural Basis for the Stromal Edema in Klf4CN Mouse Corneas. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3925.
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Klf4 is a transcription factor, highly expressed in cornea, with an important role in maturation and maintenance of the adult ocular surface. Here we examined the structure of corneal stroma in KlfCN mice with selective deletion of the Klf4 gene, where corneal expression of the gene was abolished, in order to characterize the stromal edema previously reported in these animals.
Corneas together with a rim of sclera were removed from wild-type and KlfCN mouse eyes and transferred to 4% paraformaldehyde fixative in 0.1M phosphate buffer. Small angle X-ray scattering was carried out at the UK Synchrotron Radiation Source, Daresbury with a beam approximately 0.5mm square. Corneas were enveloped in plastic film to prevent dehydration and the beam passed through full tissue thickness along the optical axis. Exposures were made along a vertical meridian at 0.5mm intervals using a computer-operated translation stage. Patterns were analyzed to produce scattering intensity plots from which collagen fibril Bragg spacing was calculated. Some corneas of wild-type and KlfCN mice were then removed to fixative -/+ Cuprolinic blue, embedded in epoxy resin and sectioned for routine electron microscopy and proteoglycan localisation, respectively.
An average collagen interfibrillar Bragg spacing of 44.5nm (standard deviation +/- 1.8nm) was calculated for collagen fibrils in central cornea of wild type mice (n = 5), compared to 66.1nm (standard deviation +/- 1.8nm) in KlfCN mice (n = 8) with selective deletion of the Klf4 transcription factor gene. Consistent with the X-ray scattering data, electron microscopy illustrated increased fibril spacing at locations in anterior, mid and posterior stroma of the central cornea of KlfCN mice compared to wild type. Images also suggested an increase in fibril diameter throughout the tissue. Specimens fixed in the presence of Cuprolinic blue showed an apparent reduction in proteoglycan-dye complexes.
Corneal edema in mice with selective deletion of Klf4 expression was reflected at the ultrastructural level in markedly increased collagen interfibrillar spacing throughout the full thickness of the central stroma, perhaps accompanied by changes in fibril diameter and proteoglycan associations.
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