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Emily Davis, Kelli Theisen, George McArdle, Rebecca Zoltoski; Using EdU in whole pig lenses to establish anterior epithelial cell division rates. Invest. Ophthalmol. Vis. Sci. 2013;54(15):473. doi: https://doi.org/.
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We are currently investigating methods to establish the location and mitotic rate of germinative zone lens epithelium (GZ LE) in whole pig lenses. We want to determine the percentage of GZ LE cells that are in the mitotic state by applying the DNA marker, 5-ethynyl-2'-deoxyuridine (EdU). The methods established in this project will ultimately help assess changes due to laser ablation targeting these cells as a potential presbyopia treatment.
Fresh pig eyes were obtained from a local abattoir. Whole lenses were treated chymotrypsin to remove ciliary body and then exposed to 0.5 mM EdU for 2 hours. Samples were fixed and permeabilized, and then EdU was visualized using Click-iT Alexa 488 or 594 fluorescence replication marker. The whole lenses were then secured on a stage so that the anterior surface was flat and pictures were obtained using a Nikon AR1 multiphoton microscope. Some lenses were counter stained with Hoechst 33342 to visualize nuclei.
Approximately 30-40 GZ LE cells were labeled with EdU, located approximately 500 µm from the lens equator. We would expect that the lens growth rate has started to slow in a 6-month-old pig, so this rate appears to be appropriate for that age range.
This method will allow us to establish germinative zone location and size in a whole lens, which will permit accurate identification of these cells in future applications. These methods will assist our lab in in our studies utilizing laser ablation of GZ LE.
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