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Ayman J Aljohani, Shari A Seidman, Natasha A Johnson, Ulises Arbelo, Katyayini Aribindi, Sanjoy K Bhattacharya; Corneal protein and lipid alterations in response to metallic impaction. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4330. doi: https://doi.org/.
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Tissue response to metallic contact and injury is not well understood. Our project aims to determine the effect of metallic object impaction on protein in the corneal tissue. We utilized bovine, porcine and human corneas in these experiments.
The human cornea samples were collected adhering to tenets of declaration of Helsinki under approved IRB or exempted protocols. We used corneas from bovine, porcine (n=300 each), and human donors (n=6) to characterize proteomic and lipidomic profile after metallic exposure (impaction with iron, copper, and lead). All corneas were carefully evaluated by Fluorescein staining for epithelial integrity. Metallic objects were used to penetrate corneal cross sections (3-4 mm) at various depths. The cornea was then cut into equal pieces with one piece serving as control. Corneal pieces were incubated for predetermined time intervals (30 min, 1 hour, 4 hours, 6 hours and 24 hours) with the retained metallic objects in place to simulate real-life situations. At the end of the designated time period, the metallic object was removed and proteins were extracted using Bradford’s assay method. Post fractionation and protease digestion, protein identification was carried out using a Q-exactive and class specific lipid identification using a TSQ Quantum Access Max mass spectrometer. During LC-MS/MS analysis all spectra were searched using Swiss Prot database. All results were subjected to statistical analysis.
Iron impaction to corneal tissue resulted in cleavage of 1-phosphatidylinositol-4, 5-bisphosphate phosphodiesterase beta-2 variant (PLCB2; 134KDa) into a 36 kDa species. Penetration of the cornea with other metals (copper and lead) resulted in lower protein extractability from corneal tissue compared to controls but not distinct changes in PLCB2. Iron impaction of corneal tissue for 24 hours resulted in cleavage of PLCB2 commensurate with significant changes in phosphatidylinositols (PIs) but not phosphatidylcholines (PCs) or other phospholipids.
The changes in protein profiles were unique to the type of metal used for impaction. The depth of injury negatively affected protein extractability and profile compared to controls. Significant changes in phosphatidylinositol were observed with iron impaction but not with copper or lead.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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