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Rachida Bouhenni, Jeffrey Dunmire, Ying Liu, Qundeel Rafiq, David Gothard, James King, Melissa Naiman, Rafat Ansari, Deepak P Edward; Identification of Molecular Signatures in Vitreous Humor Following Laser Exposure using Dynamic Light Scattering. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4630.
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© ARVO (1962-2015); The Authors (2016-present)
To identify signal changes in the vitreous humor (VH) using dynamic light scattering (DLS) and proteomic analysis following laser induced retinal injury in an animal model.
Three grades of retinal laser lesions were applied using a continuous 532 nm laser, producing minimally visible lesions (MVL), grade two (GII) or grade three (GIII) in one eye of each animal (n=4/group; 50 lesions per eye). DLS measurements were performed in vivo before and at 4hrs, 24hrs and seven days after laser treatments in control and laser treated eyes using a customized instrument. VH samples collected from enucleated eyes were analyzed by Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS) and relative protein abundances were determined by spectral counting. Significance in the differential expression of proteins between treated and untreated groups was determined by G-test followed by post-hoc Holm Sidak. For DLS, a double exponential distribution was employed to model the diameter and intensity of small and large particles at depth locations increasing by 0.1 mm increments along the optical axis of the eye. For each group, a moving range of 5 peripheral measurements corresponding to a 0.5 mm range were performed to account for anatomical variation in determining significant changes from pre-treatment measurements in the mean ratio of smaller particles average diameter to intensity.
DLS signal analysis of treated VH samples, revealed significant changes in particle diameter and intensity in the GII treated samples at 24hrs. Changes were detected at 10-11 mm posterior to the cornea. Differences in protein profile in the VH of the laser treated eyes were noted when compared to control. Some of the proteins that were significantly upregulated included carbonic anhydrase 3 (5.3fold, p=0.0001), alpha crystallin B chain (3.3fold, p=0.0001) and beta crystallin B2 (2.99fold, p=0.00013).
DLS signal and protein changes were detectable in VH following retinal laser injury. This suggests that laser exposure induces upregulation of proteins in the retina that leak into the VH from the damaged tissue. These proteins can be detected non-invasively using DLS and may be used as biomarkers for laser induced retinal injuries in field applications.
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