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. 2013 Aug 8;8(8):e72135.
doi: 10.1371/journal.pone.0072135. eCollection 2013.

670nm photobiomodulation as a novel protection against retinopathy of prematurity: evidence from oxygen induced retinopathy models

Riccardo Natoli  1 , Krisztina Valter, Marconi Barbosa, Jane Dahlstrom, Matt Rutar, Alison Kent, Jan Provis
Affiliations

670nm photobiomodulation as a novel protection against retinopathy of prematurity: evidence from oxygen induced retinopathy models

Riccardo Natoli et al. PLoS One. .

Abstract

Introduction: To investigate the validity of using 670nm red light as a preventative treatment for Retinopathy of Prematurity in two animal models of oxygen-induced retinopathy (OIR).

Materials and methods: During and post exposure to hyperoxia, C57BL/6J mice or Sprague-Dawley rats were exposed to 670 nm light for 3 minutes a day (9J/cm²). Whole mounted retinas were investigated for evidence of vascular abnormalities, while sections of neural retina were used to quantify levels of cell death using the TUNEL technique. Organs were removed, weighed and independent histopathology examination performed.

Results: 670 nm light reduced neovascularisation, vaso-obliteration and abnormal peripheral branching patterns of retinal vessels in OIR. The neural retina was also protected against OIR by 670 nm light exposure. OIR-exposed animals had severe lung pathology, including haemorrhage and oedema, that was significantly reduced in 670 nm+OIR light-exposed animals. There were no significance differences in the organ weights of animals in the 670 nm light-exposed animals, and no adverse effects of exposure to 670 nm light were detected.

Discussion: Low levels of exposure to 670 nm light protects against OIR and lung damage associated with exposure to high levels of oxygen, and may prove to be a non-invasive and inexpensive preventative treatment for ROP and chronic lung disease associated with prematurity.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
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.
Figure 2
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.
Figure 3
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).
Figure 4
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).
Figure 5
Figure 5. Analysis of peripheral vessel branching patterns in the mouse OIR model.
The hierarchical clustering diagram indicates a divergence from the experimental groups with the largest divergence occurring with the OIR group. The Mahalanobis distance was calculated from a MANOVA procedure and is shown relative to control. The OIR group clusters alone (1.96 distance) indicate a very different pattern of peripheral vasculature, to the other experimental groups. 670nm+OIR (0.46 distance) reduced the alteration in the peripheral patterning to almost that of control and 670nm (0.32 distance). The length of the lines in the hierarchical clustering diagram indicate the relative difference between groups.
Figure 6
Figure 6. Number of retinal haemorrhages in the rat oxygen induced retinopathy animals.
The number of haemorrhages was reduced from 2.3 per retina in the OIR animals to 0.4 in the 670nm+OIR animals. The 670nm+OIR was not statistically significant from controls, but was from OIR animals (* indicates a p-value < 0.05). OIR – Oxygen Induced Retinopathy. Graph bar fills indicate OIR (light grey) and 670nm+OIR (white).
Figure 7
Figure 7. Animal weight (A), length (B), mortality rate (C) and organ weight (D) taken daily in animals exposed to 670nm compared to controls in normal atmospheric oxygen.
(A) From P12 there was a statistically significant (* indicated p<0.05) deviation from normal weight gain with animals gaining weight at an increased rate. (B) There were no noticeable differences in the length of the animals over the 21 day period. (C) There was no change in the weight of the organs investigated except for the lungs which showed a significant increase in weight (* indicates p < 0.05). (D) 670nm red light appears to reduce the mortality rate from 27% in controls (n=27) to 15% in the 670nm (n=21) exposed animals (* indicates p<0.05). All animals were raised in normal atmospheric oxygen. Littermates were used to reduce variability across litters. Graph bar fills indicate controls (grey bars), 670nm treated (white bars).
Figure 8
Figure 8. Assessment of the organ weights and lung pathology of rats exposed to either OIR, 670nm or both.
There was no change in the weights of animals exposed to 670nm from control (A). The OIR animals showed a statistically significant decrease in organ weight in both the kidneys and liver (* indicates p <0.05). There was an increase in the number of lung pathology (B) presentations (both haemorrhages and oedema) in the OIR exposed animals. This was ameliorated by 670nm exposure compared to control levels. 670nm alone showed no change from control levels. 670nm treated OIR animals did not show any difference from OIR levels. OIR – Oxygen Induced Retinopathy. Graph bar fills indicate controls (black), 670nm treated (dark grey), OIR (light grey) and 670nm+OIR (white).
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Australian Research Council Centres of Excellence Program Grant (CE0561903); Canberra Hospital Private Practice Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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