An ultrastructural, time-resolved study of freezing in the corneal stroma.

Synchrotron X-ray diffraction was used to monitor the changes occurring in the extracellular matrix of the corneal stroma as a result of freezing and thawing. The parameters monitored were the lateral centre-to-centre spacing between the collagen molecules within the fibrils (intermolecular spacing) and the centre-to-centre spacing between the collagen fibrils (interfibrillar spacing). Our findings suggest that, while frozen, the fibrils are reduced in diameter and are forced into close association with each other. The data also suggest that the extrafibrillar components of the cornea may become concentrated around the fibrils during freezing. However, X-ray patterns of thawed corneas show normal interfibrillar and intermolecular spacings. Time-resolved data show that, as thawing takes place, the fibrils gradually separate and regain their normal spacing while at the same time regaining their normal diameter. It seems probable that the mechanism which allows the fibrils to regain their normal arrangement after thawing involves charge interactions between the proteoglycans associated with the fibrils. However, unlike corneas at physiological hydration, certain regions of the stroma of swollen corneas do suffer irreversible damage as a result of freezing. It is possible that this ice damage may occur in regions of abnormal fibril arrangement called "lakes", which are reported to occur in swollen cornea.