Discipline: Biological Sciences
Kenneth Terry II - University of New Orleans
The cornea is the outermost tissue of the eye, characterized by its transparency and dense innervation mainly by sensory nerves. It is also protective barrier against bacteria, viruses, and any other type of foreign object or particle from entering the ocular sphere. Inflammation is the body?s natural response against trauma, barrier break or microbial infection, but delayed resolution of inflammation can damage the tissue with severe consequences for vision. Previous studies from our laboratory have found that topical treatment of PEDF plus DHA after corneal injury stimulates nerve regeneration and decreases the inflammatory response. The molecular mechanism of their action involve the synthesis of a lipid with a fragmentation pattern by LC-MS/MS similar to Resolvin D6 (RvD6), but with a retention time different that the standard of RvD6, suspecting that we are in the presence of an isomer that have bioactive properties. Specialized pro-resolving mediators (SPMs) of inflammation, such as RvD6 are important to control inflammation. The objective was to further identify the synthesis of RvD6.
Using mice as our in vivo model allowed an efficient yet effective way of visualizing and testing the inflammatory response. Mice were handled with guidelines of the ARVO statement for the use of Animals in Ophthalmology and Vision Research. Male CD-1 mice were injured using 2.0 mm trephine to the center of the cornea that damage the corneal nerves and induce an inflammatory response. Mice were euthanized 20-hours post-injury and the corneas were incubated in the presence of inhibitors for 5-LOX (MK591 100uM), 15-LOX (ML351 200uM), Fluvoxamine (P450 200uM), or 17-ODYA (P450 50uM) with PEDF+DHA for 4 hours. The medium was then collected and the lipids were extracted by Blight and Dyer method. Each inhibitor was chosen because of the known pathways of DHA. To determine if the relative amounts of RvD6 is actually coming from the PEDF+DHA sample and not the host membrane, we used DHA Deuterium isotope(DHAd5) plus PEDF to demonstrate that the RvD6 is synthesized from the DHA we added. Lipids were analyzed by liquid chromatography-tandem mass spectroscopy (LC-MS/MS). RvD6 was identified with the full fragmentation chromatogram and UV spectrum. Results were noted and compared to the internal standard (LTB4-D4). The Fluvoxamine inhibitor showed the largest inhibition of RvD6, with further research it may be concluded that the P450 produce an isomer of RvD6.
Funder Acknowledgement(s): National Science Foundation ; National Institute of Health
Faculty Advisor: Ashok Puri, email@example.com
Role: I participated in the inhibitor incubation process, lipid extraction, data analysis, and mice euthanization.