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2 International Journal of Photoenergy In this study we used the light-induced rodent model of retinal degeneration to investigate the dose effects of 670 nm light on retinal damage in vivo. We also investigated the direct effects of 670 nm light on mitochondrial function in an immortalized line of photoreceptor (661 W) cells. We found that the dose of 670 nm light needed for a therapeutic effect varies with severity of retinal damage and that excessive exposure to 670 nm light can result in retinal damage. We also found that 670nm light directly influences mitochondrial metabolism by increasing the spare respiratory capacity of mitochondria in 661W cells adding further evidence of mitochondrial involvement in photobiomodulation. 2. Methods 2.1. Animal Rearing. All animal procedures were conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research, as well as The Australian National University (ANU) Animal Experimentation Ethics Committee (ANU Ethics Protocol Number: A2011/029). Ani- mals used for these experiments were adult albino Sprague- Dawley (SD) rats aged between 90 and 120 days. The animals were born and raised in a 12-hour light, 12-hour dark cycle (12 : 12 L/D) in dim light conditions (5 lux). 2.2. 670 nm Light Treatment and Light Damage. Treatment with 670nm light was conducted using a WARP 75 light source (Quantum Devices Inc., Barneveld, WI) a light emitting diode (LED) array producing a power flux of 60 mW/cm2 . 670 nm light treatments commenced consis- tently at 9:00 AM every morning. Animals were individually towel-wrapped to aid in handling during treatments. The light source was positioned approximately 2.5 cm away from the eyes of the animal when treated. Animals received one of the following doses of 670 nm light: 9 J/cm2 (3-minute continuous treatment); 18 J/cm2 (6-minute continuous treat- ment); 36 J/cm2 (12-minute continuous treatment); 90 J/cm2 (30-minute continuous treatment). Animals were treated once daily on five consecutive days. Immediately following the fifth day of 670 nm light treat- ment, animals were placed in individual transparent cages. Animals were exposed for a period of 24 hours to 750-lux (“low” intensity), 1000-lux (“moderate” intensity), or 1500-lux (“high” intensity) white light, as indicated by a digital light meter placed on the cage floor (Digitech, QM1587). Light was generated from cold-white fluorescent bulbs (COLDF2 2 × 36 W IHF, THORN lighting). Control animals were exposed to white light at “low,” “moderate,” or “high” intensity, with no prior treatment with 670 nm light. 2.3. Tissue Collection and Processing. Animals were eutha- nized with CO2 immediately after light damage (LD). A mark was made on the superior surface of the eye before extraction for orientation purposes. Whole eyes were immediately injected with 4% paraformaldehyde before being immersion fixed in 2 mL of 4% paraformaldehyde solution for 4 hours at 4∘C and then processed as described previously [32]. Eyes were sectioned in the sagittal plane at a thickness of 16 𝜇m and mounted on poly-L-lysine slides (Thermo Scientific, Waltham, MA). 2.4. Histological Analyses 2.4.1. Cell Death. 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 [32] using a fluorophore, Alexa 594, for visualization. Counting was done manually by a skilled investigator, who was “blind” to the treatment protocol, using a Zeiss AxioVert 200 (Zeiss Aus- tralia, North Ryde, NSW) inverted fluorescence microscope. The retina was divided into eight equal-sized regions, four on the superior and four on the inferior. TUNEL+ cells in the ONL (Outer Nuclear Layer) of the retina were counted in each region; then all counts were added together to give the total amount of TUNEL+ cells in the retina for that respective eye section. Representative TUNEL images were taken on a Nikon A1 Confocal microscope (Nikon, Tokyo, JP). 2.4.2. ONL Thickness Ratio. ONL ratios were analyzed on a Zeiss AxioVert 200 inverted fluorescence microscope. The ONL contains the cell bodies of the photoreceptors and will become thinner as more layers of photoreceptors die through apoptosis, providing a measure of retinal degeneration. The thickness of the ONL layer was measured using ProgRes Capture Pro (Jenoptik AG, Jena, Germany) and divided by the thickness of the overall retina spanning from the inner limiting membrane to the outer limiting membrane in order to generate the ONL thickness ratio. One measurement was taken from each of the eight equal-sized regions of the retina before being averaged together. 2.5. Cell Culture. The 661W photoreceptor cell line used in these studies was generously provided by Dr. Muayyad Al-Ubaidi (University of Oklahoma). Cells for experimental purposes were used within 5 passages of authentication. Val- idation of the cells authenticity was performed by Cell Bank Australia for species specificity. They were further validated using RT-PCR for expression of cell specific markers includ- ing cone arrestin, opsin pigments, and SV40. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), L-glutamine (3 mM), sodium pyruvate (1 mM), and glucose (25 mM). Cells were allowed to grow to 80–90% confluence before use for experiments. Cells were kept in 37∘C and 5% CO2. Cells treated with 670 nm light were given two separate 9J/cm2 (3-minute) treatments before the running of the respective assay. Cells were treated once before light damage, and once directly after. Treatments were conducted using a WARP 75 light source (Quantum Devices Inc., Barneveld, WI) LED array at 60 mW/cm2 energy flow emitting light at a wavelength of 670 nm. The LED was placed underneath the cell culture plate so that the cells were directly subjected to the light source.PDF Image | 670 nm Light Therapy to Protect vs Photoreceptor Cell Death
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