Structural Studies of the Corneal Stroma

Goodfellow, Julia Mary (1975). Structural Studies of the Corneal Stroma. PhD thesis The Open University.



The corneal stroma is an unusual connective tissue in that it is transparent to visible light and that it can swell to many times its original weight when placed in salt solution with a consequent loss in transparency with swelling. The stroma consists of lamellae of parallel collagen fibrils, of uniform diameter, embedded in a ground substance. It also contains glycosaminoglycans which are negatively charged at physiological pH. The organisation of the collagen fibrils and their relationship to the glycosaminoglycans are important when considering both the transparency and the ability to swell of the fresh tissue and the reason for the loss in transparency on swelling.

Experiments were undertaken to study changes in the stroma which take place on swelling as a function of the pH and~he ionic strength of the bathing solution. Initially, the total water content per unit dry weight is studied as a function of the bathing solution and the time of dwelling. Low-angle x-ray diffraction techniques are used to monitor the centre-to-centre distance between the collagen fibrils (the interfilament spacing) as a function of hydration as well as the reflections due to the packing of the tropocollagen molecules from which the fibrils are formed. Thirdly, the fixed charge concentration due to the polyelectrolytes in the stroma is calculated from microelectrode measurements of the potential difference which exists between the stroma and the bathing solution. Fourthly; the absorbance of the stroma to visible light is measured as a function of hydration in various solutions.

The results of these four different techniques indicate the importance of the fixed charge on the corneal macromolecules. This fixed charge must give rise to an unequal distribution of permeant ions between the stroma and the bathing solution which leads to a Donnan, osmotic, pressure. This Donnan pressure appears to be the main cause of swelling. Measurement of the fixed charge from the potential difference which also arises from the unequal distribution of permeant ions, shows similar behaviour with pH and ionic strength to that estimated from the swelling results. This dependence is also similar to the behaviour expected from polyelectrolyte gel theory. The value of the interfilament spacing, for a given hydration, is shown to depend on the pH of the bathing solution so that more fluid goes into the lattice of fibrils with some solutions that with others. The water not going into the lattice may be in the form of 'lakes' as suggested by Benedek (1971). The reflections from the packing of tropocollagen molecules along the fibrils are unusual in that, although they index on a repeat of around 66nm, the first order reflection is absent. The Patterson function from such a x-ray pattern is calculated and compared with that from scleral collagen which shows the first order reflections. Finally, the transmission of light by the stroma is shown to decrease linearly with increasing hydration, the rate of decrease being dependent on the pH of the bathing solution. Correlations between the rate of loss of transmission and the rate of swelling as well as between the percentage water in 'lakes' with the transmission of the stroma are discussed.

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