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Ravin Sajnani, Genea Edwards, Sanjoy K Bhattacharya; Evaluation of transport time of lipids in anterior segment organ cultures fitted in 3D-printed holding structures. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1069.
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Bioactive lipids could be potential modulators of cellular regulatory processes. We attempted to determine the transport time of selected lipids in the anterior segment angle region using anterior segment explants fixated on 3D-printed holding structures.
Human anterior segment explants (n = 5 each male and female, all Caucasian, age 60±15 years) were prepared following established methods from non-glaucomatous cadaveric donor eyes free from apparent corneal defect. Phosphatidylcholine (m/z 649.90), phosphatidylserine (m/z 810.03), and sphinganine (m/z 301.52) lipid standards with or without fluorescent head group modification were procured (from Avanti Polar Lipids and Echelon Biosciences). Autodesk Fusion360 software was used to create a 3D holding structure. Transport time was calculated after topical application of lipids on the corneal surface and sampling from ports located at the pseudo-iridocorneal angle at various time points. We term the pseudo-iridocorneal angle to describe the location which will be near equivalent to iridocorneal angle in the eye in vivo. The samples were subjected to lipid extraction, identified, and quantified using mass spectrometry. The fluorescent analogs also allowed parallel quantification using fluorescent measurement from the same samples prior to mass spectrometric analyses.
The identification of lipids necessitated lipid extraction. Direct identification of lipids without lipid extraction was erroneous. We found little difference in the transport of phosphatidylcholine (PC) with or without a vehicle. Fluorescent analogs of phosphatidylserine took slightly longer time than PC to reach the pseudo-iridocorneal angle. We found 35-80 percent of applied PC lipids reach the pseudo-iridocorneal angle in anterior segment cultures when used topically in our model.
Topical application of phospholipids results in their transport to the pseudo-iridocorneal angle. The 3D structure provides a good system to hold the anterior segment in a desirable manner for evaluation of the transport of lipids and their fluorescent analogs.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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