CONTROL OF CORNEAL HYDRATION

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1. It is proposed that a macromolecular solute in either the aqueous humor in the stromal tissue fluid, but absent from tissue culture media, is responsible for the functional and structural well being of the endothelial cells of the cornea in their natural environment. This will be tested in the living eye by inserting permeable membranes with different pore sizes between the cell layer and the media on either side. Both the return of function of tissue cultured cells replaced in the eye and the loss of function of native cells will be looked for. 2. The technique of suturing tissue-cultured layers of endothelial cells onto the rear surface of the cornea as an alternative to corneal grafting will be evaluated. 3. The permeability of the limiting cell membranes of the endothelial cells to ions will be measured with a view to identifying which ones are involved in the fluid pump mechanism. Radioisotope solutions will be brought into contact with the cell layer from either side, and then it will be quick frozen, freeze dried and collected to measure how much tracer penetrated. Comparison of the membrane permeabilities will be made when the cell layer is pumping and when inhibited. 4. The role of the bicarbonate and hydrogen ion concentration gradients across the cornea on the pump activity of the endothelium will be tested in vitro by adjusting the pH at the media on either face of the tissue at a constant CO2 partial pressure. 5. The possibility that the transport or the leakage of water across the endothelial cell layer takes place through the intercellular junctions will be explored by looking for microcurrents in the fluid adjoining the cells. These will be identified by observing the displacement of oil droplets suspended in the bathing fluid. 6. The patterns of growth of blood vessels invading the stroma will be observed in order to see if growth is regulated by the ultrastructure of the tissue and whether the formation of new vascular loops is by the chance encounter of endothelial sprouts or is directed by chemical or structural stimuli.