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weight infants [9]. Use of these guidelines significantly reduces the risk of blindness and significant visual loss, but does not prevent the disease from occurring, nor the other long-term visual consequences of ROP. Current therapies for ROP, including laser photocoagulation or cryo-therapy to reduce the incidence of blindness, although a number of long-term visual issues can still present post treatment including reduced visual acuity [10], reduced visual fields [11], reduced contrast sensitivity [12] and strabismus [13]. All current treatments for ROP are invasive, expensive and target only the angiogenic aspect of the disease, thus not addressing the neuronal impacts or other long-term effects. Thus there is a need for an inexpensive, non-invasive preventative treatment for ROP. Low-level Light therapy (LLLT) in the red to near-infrared light spectrum (600-1000nm) protects against neuronal and retinal cell damage [14], and 670nm red light has been shown to be a powerful neuroprotectant, against light induced damage [15,16] and toxins [17]. Treatment with 670nm red light improves retinal healing [18] and modulates expression of genes involved with inflammation, oxidative metabolism and apoptosis [19]. Although the precise mechanism is unknown there is strong evidence to suggest that cytochrome c oxidase (CCO) acts as the primary photo-acceptor/chromophore [20], boosting oxidative metabolism [21] and ATP production [22], driving reparative and protective mechanisms. Animal models for ROP are well established and include a number of rodent models. These oxygen-induced retinopathy (OIR) models have provided crucial insights into the pathogenesis and underlying mechanism of ROP [23]. OIR models take advantage of the fact that in some animals normal retinal vascularisation occurs ex utero, and thus resemble the incomplete vascular development of the retinal vasculature in premature infants. The aims of the present study were to use mouse and rat models to evaluate the efficacy of 670nm red light to protect against OIR. We hypothesise that treatment with 670nm red light will be a novel preventative strategy for ROP, promoting normal mechanisms of retinal vascularisation, reducing oxidative and neuronal damage. Materials and Methods Animals All work was conducted using either C57BL/6J mice or Sprague-Dawley albino rats. All animal experimentation was conducted in accordance to the ARVO (Association for Research in Vision and Ophthalmology) statement for the Use of Animals in Ophthalmic and Vision Research and with the approval of the Animal Ethics Committee at the Australian National University, Canberra (protocol-A2011/029). Animals were raised and experiments conducted in cyclic 5 lux light (12hrs: 12hrs). All animals were culled using cervical dislocation. All culling was performed at 9am to control for possible circadian effects. For both the mouse and rat OIR models animals were assigned to one of 4 groups; control (normal oxygen, no 670nm), 670nm (normal oxygen, 670nm treatment only), OIR (oxygen only, no 670nm treatment) or 670nm+OIR (oxygen and 670nm treatment). To adjust for litter size and cross-litter variability all litters were maintained at 10 animals per experimental group and 2 experimental groups per cage/dam (control and 670nm, or OIR and 670nm + OIR). All experiments were performed in biological triplicate (3 litters per experimental group), with a minimum of 12 animals per experimental group. Oxygen Induced Retinopathy Models Mouse 75% oxygen model. Animals were maintained in normoxia until P7. At P7 dam and pups were placed in an Oxycycler (Biospherix Ltd., Lacona, NY) with constant 24hrs 75% oxygen for 5 days. At p12 animals were removed from oxygen and returned to normoxia until P17. At P17 animals were culled and the eyes and organs harvested for further investigation. Animal weights and lengths were measured daily at 9am. Animals weighing less than 4g at P17 were excluded from the study. Rat 80%/21% oxygen model. At birth (P0) pups and dam were placed in an Oxycycler (Biospherix Ltd., Lacona, NY) with 24 hours cyclic oxygen, 80% for 22 hours: 21% (normoxia) for 2 hours. Animals were maintained this way until P18 when they were culled, and the eyes and organs harvested for further investigation. Animal weights and lengths were measured daily. Animals weighing less than 30g at P18 were excluded from the study. 670nm Red Light Treatment Treated animals were exposed to 670nm red light only during exposure to hyperoxia (P7-P17, mouse; P0 – P18, rat). Animals were exposed to 670nm red light from a WARP 75 source (Quantum Devices Inc., Barneveld, WI) Each animal was held approximately 2.5cm from the light source and treated for 3 minutes daily. This arrangement provided a fluence of 9J/cm2 at the eye. The animals did not appear agitated by the red light. All treatments were performed at 9am. Retinal Whole-mounts Retinal whole-mounts were prepared using an established technique [24] with slight modifications. Eyes were removed and immediately fixed in 4% paraformaldehyde for 1 hour. Following fixation the eyes were rinsed 3 times in phosphate buffered saline (PBS). The retinas were removed from the eyecup, and placed under a dissection microscope (Leica, Wetzlar, Germany). To whole-mount the retina an ophthalmic surgical blade was used to create four incisions 1mm from the optic nerve head to the peripheral retina. Retinas were fixed in 4% paraformaldehyde for four hours then washed overnight in PBS. Retinas were then washed with PBS for three intervals of 30 minutes and placed in a fluorescein isothiocyanate (FITC) conjugated lectin stain (Sigma, St. Louis, MO) at a ratio of 1:100 with PBS for 24 hours, followed by rinsing with PBS for three intervals of 30 minutes. Retinas were then whole- mounted on slides and visualised using LSM 5 confocal microscope (Carl Zeiss, Jena, Germany) and acquired using PASCAL v 4.0 software (Carl Zeiss). Individual images were stitched together to create whole retinal images and prepared for publication using Adobe Photoshop CS4. Assessment of PLOS ONE | www.plosone.org 2 August 2013 | Volume 8 | Issue 8 | e72135 670nm Protects against Retinopathy of PrematurityPDF Image | 670nm Photobiomodulation as a Novel Protection to Retinopathy
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