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Svein Estil, Earl J. Primo, Graeme Wilson; Apoptosis in Shed Human Corneal Cells. Invest. Ophthalmol. Vis. Sci. 2000;41(11):3360-3364.
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purpose. 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
conclusions. 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
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