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doi: 10.1371/journal.pone.0072135.g002 Figure 3. Quantification of vascular abnormalities in P17 c57BL/6J mouse lectin stained retinal wholmounts. (A, B) OIR shows increased vaso-obliteration (E) and neovascularisation (F) compared to 670nm + OIR (C, D). Neovascularisation was reduced to almost control levels in the 670nm+OIR retina (F). Red (A, C) areas show vaso- obliteration, while white arrows show large neovascular tuffs and white arrows show small tufts. * indicated statistical significance p<0.05. Error bars show standard error. P – postnatal day, OIR – Oxygen induced retinopathy. Graph bar fills indicate controls (black), 670nm treated (dark grey), OIR (light grey) and 670nm+OIR (white). doi: 10.1371/journal.pone.0072135.g003 (Figure 1C,G, Figure 2C,G). Analysis of the retinal peripheral vasculature branching patterns showed that OIR retinas had a distinctive pattern of branching, compared to controls. The cladistic analysis (Figure 5) indicates that OIR retinas clustered Figure 4. Quantification of vaso-obliteration and cell death in P18 Sprague-Dawley rats. (A) OIR shows increased vaso- obliteration (C) compared to 670nm + OIR (B). Red (A, B) areas show vaso-obliteration. Representative images (D–E) and quantification (H) of TUNEL images from the rat retina. To maintain consistency representative images (D–E) were all taken 500μm from the optic nerve head on the superior side of the retina. Arrows indicate positive cell labelling in both the OIR and 670nm+OIR animals. Control and 670nm (dark showed little TUNEL positive labelling (H) in any of the retinal layers, while experimental groups exposed to OIR showed increased labelling compared to control levels (H). 670nm+OIR reduced the level of labelling from that of OIR, but only with statistical significance in the ONL (H). * indicated statistical significance p <0.05. Error bars show standard error. Arrows indicate positive cell labelling. GCL – ganglion cell layer, INL – Inner nuclear layer, ONL – outer nuclear layer, P – postnatal day, OIR - Oxygen Induced Retinopathy. Scale A,B = 1mm, D-G = 50μm. Graph bar fills indicate controls (black), 670nm treated (dark grey), OIR (light grey) and 670nm+OIR (white). doi: 10.1371/journal.pone.0072135.g004 670nm Protects against Retinopathy of Prematurity Figure 2. P18 Sprauge-Dawley rat retinal wholemounts, stained with lectin. (A,E) Control and (B,F) 670nm alone mice retina showed no observable difference in peripheral retinal vasculature, while (C,G) OIR alone showed increaed vaso-obliteration (indicated by +) and areas of retinal heamorrhages (indicated by an arrow). (D,H) 670nm+OIR showed amelioration of vaso- obliteration and control like vascular patterning. P – postnatal day, OIR - Oxygen induced retinopathy. Scale A-D = 2mm, E-H = 650μm. Magnification of images E-H are 4x original images. PLOS ONE | www.plosone.org 4 August 2013 | Volume 8 | Issue 8 | e72135PDF Image | 670nm Photobiomodulation as a Novel Protection to Retinopathy
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