Apoptosis in shed human corneal cells

To determine whether shear forces applied to the corneal epithelium by the repeated insertion and removal of a hydrogel contact lens alter the size and number of cells removed and to determine the contribution of apoptosis to this process. METHODS; Human corneal cells were collected from eight healthy subjects by sequential contact lens cytology (20 lens insertions and removals). Collected cells were stained with acridine orange for counting and measurement of cell size. In a separate experiment, collected cells were fixed and stained with TdT-mediated dUTP nick-end labeling (TUNEL) or labeled immediately after collection using annexin V. Hoechst stain and propidium iodide (PI) were used as nuclear counterstains. The proportion of cells labeled with acridine orange, TUNEL, and annexin V was quantified by fluorescence microscopy. RESULTS; The number of cells increased in later collections, and cells were smaller. The mean number of positively stained cells using TUNEL was 57%. Annexin V labeling on unfixed fresh samples showed a mean of 64%, with an increase in later collections. Apoptotic bodies were observed in very few cells. In most cells the nucleus and cytoplasmic membrane were intact. Structures were observed in which nuclei were missing (Hoechst negative) but in which cytoplasm had the size and appearance of whole, nucleated cells. These structures (cell ghosts) increased in number along with the increase in nucleated cells in later collections.

The sequential removal of a soft contact lens caused a progressive increase in the number of cells collected from the surface and a progressive decrease in their size. The majority of nucleated cells removed by a contact lens were apoptotic in the sense of being positively labeled by TUNEL and annexin V. Morphologically they differed from classically apoptotic cells, in that cells showed an intact nuclear structure and no discernible apoptotic bodies. They could represent a last stage in a pathway of cell differentiation in which frictional forces induced by the removal of the contact lens activate the apoptotic program and cause the cell to be shed. There is also a pathway in which cells lose their nuclei before leaving the epithelial surface.