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Steven Charles Quan, Taia Cordel, Rich Miller, Andy Ritter, Rebecca K Zoltoski, George McArdle; Establishing a Model to Investigate Anterior Epithelial Cell Division in Whole Pig Lenses. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2625.
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© ARVO (1962-2015); The Authors (2016-present)
We are developing a reliable method to determine the specific location and mitotic rate of germinative zone lens epithelium (GZ LE) in whole pig lenses using intact pig lenses and a DNA marker called 5-ethynyl-2'-deoxyuridine (EdU). With the ability to locate mitotic cells and measure baseline rates of cell division, we intend to manipulate laser ablation as a potential treatment for presbyopia.
Fresh pig eyes are obtained from a local abattoir and dissected to extract whole lenses. Lenses are warmed and bathed in 3mg/mL chymotrypsin to remove excess ciliary body and then exposed to 0.6mM EdU for 2 hours (note: controls without EdU are included to assess specificity). Samples are then fixed, washed, and permeabilized, followed by incubation with a fluorescent replication marker (Alexa 594) to visualize EdU uptake. Samples are counterstained with a nonspecific DNA marker (Hoechst 33342) to define cell nuclei as landmarks for co-localization with mitotic cells that have incorporated EdU. Whole lenses are secured to a stage so that the anterior lens surface is flat. Eight pictures (2 per quadrant) are taken using a Nikon AR1 multi-photon microscope. Cell quantification is completed using Fiji software. Cells are counted if maximum light intensity is at least 30% greater than that of adjacent cells with intensity representative of the target region.
The GZ of the adult pig lens is approximately 500 µM from the lens equator in a band that is roughly 500 µM wide. All experimental lenses showed staining (see table 1). The range in percentage of dividing cells per quadrant is 0 - 1.10%. The average percentage across lenses is 0.33±0.08 (SEM), while for the control lenses the range is 0- 0.01%.
This method allows us to establish the germinative zone’s location and size in a whole pig lens. As can be seen from the data per quadrant, dividing cells are not equally distributed across the anatomical GZ LE region. This staining pattern would support a concept of spiral or bidirectional development of the lens. The protocol may require additional adjustments to maximize visualization quality and yields of cell quantification. This lays the foundation for future studies utilizing laser ablation of GZ LE, with the purpose to either ameliorate and/or stop the progression of presbyopia in intact intraocular lenses.
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