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the neovascularisation and vaso-obliteration was performed as a blinded assessment using a technique described previously [24]. To quantify vaso-obliteration, lectin images were imported into Adobe Photoshop, the avascular area selected and the pixel density quantified. The number of pixels for the vaso- obliteration was normalised against the total number of pixels of the whole mount, giving a percentage of vaso-obliteration. Neovascularisation was calculated using a macro in imageJ [25] designed to specifically select areas of neo- vascularisation. The number of pixels for the neovascularisation was normalised against the total number of pixels of the whole mount, giving a percentage of neovascularisation. A one-way ANOVA with Tuckey’s post hoc test was performed using Prism 5 (GraphPad Software) to compare the effects of different treatment conditions. Peripheral Branching We used a recently submitted image processing method (Barbosa and Maddess, manuscript in preparation) namely Streamlined Morphological Image Analysis (SMIA) to characterize the change in vessel arborisation due to different treatments or conditions. SMIA features in 2D are related to known geometric measures such as the Area, Perimeter and the Euler number of a region. These quantities are monitored while the region of interest is undergoing a variational segmentation process called Chan-Vese [CV]. At critical points of this process, the most rich shape representation of the object occurs and we extract the features for SMIA, automatically. Having the SMIA geometrical features at hand we performed a one way analysis of variance (MANOVA) using their principal components in order to quantify the distance between the treatment groups. We display the result hierarchically by clustering the resulting Mahalanobis distance using a single linkage (nearest neighbour) algorithm. Organ Weights and Pathology Following cervical dislocation, animals organs were removed, weighed fresh then fixed in 10% neutral buffered formalin (NBF) and analysed for any histological abnormalities. The organs removed included the lungs, brain, kidney, liver, spleen, heart and thymus. Pathology was assessed blind and performed by an anatomical pathologist experienced in rodent histology at the Canberra Hospital, Australia. Cryosectioning and TUNEL In some animals, the fellow eye was marked on the superior aspect with a pen for orientation, enucleated and immersion- fixed in 4% paraformaldehyde for 3 hours. The eye was then washed in PBS three times and cryoprotected by immersion in 15% sucrose overnight. Eyes were sectioned at 12μm on a cryostat in the superior-inferior axis. Cell death was assessed by the TdT-mediated dUTP nick end labelling (TUNEL) technique to identify the fragmentation of DNA characteristic of apoptotic cells, following a previously published protocol [26] but using a fluorophore, Alexa 594 for visualisation. TUNEL-labelled sections were scanned from superior to inferior edge and the number of TUNEL+ profiles in Figure 1. P17 c57BL/6J mice 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 neovascularisation (indicated by arrowheads). There was also an observable vascular organisational difffernce in the OIR exposed retina (G). (D, H) 670nm+OIR, showed amelioration of neovascularisation, vaso- obliteration and a more uniform vascular patterning. P – postnatal day, OIR - Oxygen induced retinopathy. Scale A - D = 1mm, E -H = 200μm, I. Magnification of images E-H are 4x original images. doi: 10.1371/journal.pone.0072135.g001 each of the nuclear layers of the retina was recorded. Frequency of TUNEL+ profiles/mm was averaged from at least three sections per animal with 4 animals analysed. A one-way ANOVA with Tuckey’s post hoc test was performed using Prism 5 (GraphPad Software) to compare the effects of different treatment conditions. Results Effects of 670nm on Vascular Development in Oxygen Induced Retinopathy Retinal whole-mounts from mice (Figure 1) and rats (Figure 2) revealed the modifications of retinal vascular development typical of OIR paradigms, including vaso-obliteration, neovascularisation and retinal haemorrhages. In the mouse model vaso-obliteration occurs centrally, while in the rat model it occurs on the retinal periphery. In both control and ‘NIR alone’ animals, there was no adverse vascular development (n=12). Quantification of both vaso-obliteration (Figure 3 A, C, E and Figure 4 A–C) and neovascularisation (Figure 2 B, D and F) showed that in mice retinas treated with 670nm there was a significant decrease in the severity of vascular pathology (n=12, p value < 0.05). In the rat vaso-obliteration was reduced from 4.1% in OIR to 3.2% in OIR+670nm treated animals (Figure 4C). In mice vaso-obliteration was reduced from 28.9% in OIR to 20.0% (Figure 3E) in OIR+670nm animals, while neovascularisation was reduced from 6.2% to 0.9% (Figure 3F). In mice the amount of retinal neovascularisation in OIR retinas treated with 670nm was reduced to almost that of control (Figure 3F) (n=12, p value = 0.0128). The peripheral retinal vascular branching patterns in the OIR-exposed animals were anomalous, including increased tortuosity and disorganization in the peripheral vessel network 670nm Protects against Retinopathy of Prematurity PLOS ONE | www.plosone.org 3 August 2013 | Volume 8 | Issue 8 | e72135PDF Image | 670nm Photobiomodulation as a Novel Protection to Retinopathy
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