Name: method for the assessment of effects of a range of wavelengths and intensities of red/near-infrared light therapy on oxidative stress in vitro
Uploaded: 2015-03-21T15:00:00
Description: Watch this Scientific Journal Video about Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro at JoVE.com

This work was supported by the Neurotrauma Research Program (Western Australia). This project is funded through the Road Trauma Trust Account, but does not reflect views or recommendations of the Road Safety Council.

1. Optical Calibration: Measuring Light Output
<ol>
	<li>To prepare the light delivery apparatus, connect a broadband light source (e.g., Xenon or tungsten lamp) to an appropriate power supply. Position a collimating lens in front of the light source to produce a collimated beam of light. Pass the light through a liquid heat filter to remove most of the heat from the light beam. Depending on the application, focus the collimated beam on to the entrance aperture of a liquid light guide, which provides for more ...

<ol>
	<li>Gutterman, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+reactive+oxygen+species+an+evolution+in+function.">Mitochondria and reactive oxygen species an evolution in function.</a> Circulation research. 97, 302-304 (2005).</li><li>Camello-Almaraz, C., Gomez-Pinilla, P. J., Pozo, M. J., Camello, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+reactive+oxygen+species+and+Ca2++signaling.">Mitochondrial reactive oxygen species and Ca2+ signaling.</a> American Journal of Physiology-Cell Physiology. 291, C1082-C1088 (2006).</li><li>Kowaltowski, A. J., de Souza-Pinto, N. C., Castilho, R. F., Vercesi, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+reactive+oxygen+species.">Mitochondria and reactive oxygen species.</a> Free Radical Biology and Medicine. 47, 333-343 (2009).</li><li>Coyle, J. T., Puttfarcken, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress,+glutamate,+and+neurodegenerative+disorders.">Oxidative stress, glutamate, and neurodegenerative disorders.</a> Science. 262, 689-695 (1993).</li><li>Doble, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+excitotoxicity+in+neurodegenerative+disease:+implications+for+therapy.">The role of excitotoxicity in neurodegenerative disease: implications for therapy.</a> Pharmacology &amp; therapeutics. 81, 163-221 (1999).</li><li>Hall, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidant+therapies+for+acute+spinal+cord+injury.">Antioxidant therapies for acute spinal cord injury.</a> Neurotherapeutics. 8, 152-167 (2011).</li><li>Jia, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+in+spinal+cord+injury+and+antioxidant-based+intervention.">Oxidative stress in spinal cord injury and antioxidant-based intervention.</a> Spinal Cord. 50, 264-274 (2012).</li><li>Fitzgerald, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red/near-infrared+irradiation+therapy+for+treatment+of+central+nervous+system+injuries+and+disorders.">Red/near-infrared irradiation therapy for treatment of central nervous system injuries and disorders.</a> Reviews in the Neurosciences. 24, 205-226 (2013).</li><li>Rutar, M., Natoli, R., Albarracin, R., Valter, K., Provis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=670-nm+light+treatment+reduces+complement+propagation+following+retinal+degeneration.">670-nm light treatment reduces complement propagation following retinal degeneration.</a> J Neuroinflammation. 9, 257 (2012).</li><li>Eells, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+photobiomodulation+for+methanol-induced+retinal+toxicity.">Therapeutic photobiomodulation for methanol-induced retinal toxicity.</a> Proceedings of the National Academy of Sciences. 100, 3439-3444 (2003).</li><li>Begum, R., Powner, M. B., Hudson, N., Hogg, C., Jeffery, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+with+670+nm+light+up+regulates+cytochrome+C+oxidase+expression+and+reduces+inflammation+in+an+age-related+macular+degeneration+model.">Treatment with 670 nm light up regulates cytochrome C oxidase expression and reduces inflammation in an age-related macular degeneration model.</a> PloS one. 8, e57828 (2013).</li><li>Byrnes, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+promotes+regeneration+and+functional+recovery+and+alters+the+immune+response+after+spinal+cord+injury.">Light promotes regeneration and functional recovery and alters the immune response after spinal cord injury.</a> Lasers in surgery and medicine. 36, 171-185 (2005).</li><li>Liang, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobiomodulation+partially+rescues+visual+cortical+neurons+from+cyanide-induced+apoptosis.">Photobiomodulation partially rescues visual cortical neurons from cyanide-induced apoptosis.</a> Neuroscience. 139, 639-649 (2006).</li><li>Zivin, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+and+safety+of+transcranial+laser+therapy+for+acute+ischemic+stroke.">Effectiveness and safety of transcranial laser therapy for acute ischemic stroke.</a> Stroke. 40, 1359-1364 (2009).</li><li>Lapchak, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+near-infrared+laser+therapy+applied+to+promote+clinical+recovery+in+acute+and+chronic+neurodegenerative+diseases.">Transcranial near-infrared laser therapy applied to promote clinical recovery in acute and chronic neurodegenerative diseases.</a> Expert review of medical devices. 9, 71-83 (2012).</li><li>Chung, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuts+and+bolts+of+low-level+laser+(light)+therapy.">The nuts and bolts of low-level laser (light) therapy.</a> Annals of biomedical engineering. 40, 516-533 (2012).</li><li>Wu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-Level+Laser+Therapy+for+Closed-Head+Traumatic+Brain+Injury+in+Mice:+Effect+of+Different+Wavelengths.">Low-Level Laser Therapy for Closed-Head Traumatic Brain Injury in Mice: Effect of Different Wavelengths.</a> Lasers in surgery and medicine. 44, 218-226 (2012).</li><li>Hashmi, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+low-level+laser+therapy+in+neurorehabilitation.">Role of low-level laser therapy in neurorehabilitation.</a> PM&amp;R. 2, S292-S305 (2010).</li><li>Fitzgerald, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metallothionein-IIA+promotes+neurite+growth+via+the+megalin+receptor.">Metallothionein-IIA promotes neurite growth via the megalin receptor.</a> Experimental Brain Research. 183, 171-180 (2007).</li><li>Fitzgerald, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near+infrared+light+reduces+oxidative+stress+and+preserves+function+in+CNS+tissue+vulnerable+to+secondary+degeneration+following+partial+transection+of+the+optic+nerve.">Near infrared light reduces oxidative stress and preserves function in CNS tissue vulnerable to secondary degeneration following partial transection of the optic nerve.</a> Journal of neurotrauma. 27, 2107-2119 (2010).</li><li>Ando, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-level+laser+therapy+for+spinal+cord+injury+in+rats:+effects+of+polarization.">Low-level laser therapy for spinal cord injury in rats: effects of polarization.</a> Journal of biomedical. 18, 098002 (2013).</li><li>Lanzafame, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocity+of+exposure+time+and+irradiance+on+energy+density+during+photoradiation+on+wound+healing+in+a+murine+pressure+ulcer+model.">Reciprocity of exposure time and irradiance on energy density during photoradiation on wound healing in a murine pressure ulcer model.</a> Lasers in surgery and medicine. 39, 534-542 (2007).</li><li>Castano, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-level+laser+therapy+for+zymosan+induced+arthritis+in+rats:+Importance+of+illumination+time.">Low-level laser therapy for zymosan induced arthritis in rats: Importance of illumination time.</a> Lasers in surgery and medicine. 39, 543-550 (2007).</li></ol>

We have successfully adapted a precise and calibrated light delivery system to provide a mechanism for study of optimization of R/NIR-LT in vitro. Wavelength and intensity parameters of R/NIR-LT are able to be manipulated accurately and effectively using this system. We established that light treatment of the cells did not lead to cell death, although ROS were not reduced at the wavelengths and dosages delivered, in the cell types tested. The maximum intensities achieved by the current system at 670nm (20.11W/m<...

Reactive oxygen species (ROS) are required for a range of signal transduction pathways and normal reactions of cellular metabolism, including those of neuroprotection 1. However, when endogenous antioxidant mechanism are unable to control the production of ROS, cells may succumb to oxidative stress 2,3. Following injury to the CNS, the associated increases in the presence of ROS and oxidative stress are thought to play a substantial role in the progression of damage 4,5. Despite the extensive number of strategies for attenuating oxidative stress that have been assessed, there are currently no completely effective, clinically relevant anti-oxidant strategies for attenuating ROS production and associated oxidative stress in clinical use following neurotrauma 6. Therefore the attenuation of oxidative stress remains an important goal for therapeutic intervention 7.
Improvements following R/NIR-LT have been reported in a wide range of injuries and diseases including reductions in cardial infarct size, renal and hepatic complications during diabetes, retinal degeneration, CNS injury and stroke 8, perhaps by reducing oxidative stress. With particular regard to CNS injury, preclinical studies of efficacy of 670nm light have shown good effects in models of retinal degeneration 9-11, spinal cord injury 12, neuronal death 13. Clinical trials have been conducted for dry age related macular degeneration and are currently underway for stroke 14, however the outcomes of these trials do not appear promising, perhaps due to a failure to employ effective treatment parameters 15. As such, R/NIR-LT has not been widely adopted as part of normal clinical practice in neurotrauma, despite being an easy to administer, non-invasive and relatively inexpensive treatment. Barriers to clinical translation include lack of a clearly understood mechanism of action and absence of a standardized effective treatment protocol 16,17. Current literature regarding light therapy reveals a plethora of variation in treatment parameters with respect to irradiation sources (LED or laser), wavelength (e.g., 630, 670, 780, 810, 830, 880, 904nm), total dose (joules of irradiation / unit area), duration (exposure time), timing (pre- or post- insult), treatment frequency and mode of delivery (pulse or continuous) 8. The variability in treatment parameters between studies makes comparison difficult and has contributed to skepticism regarding efficacy 16.
Therefore, optimization of R/NIR-LT is clearly required, with cell culture systems able to provide the high-throughput screening mechanism necessary to compare the multiple variables. However there are few commercially available illumination systems that can provide sufficient flexibility and control over wavelength and intensity to perform such optimization experiments. Commercially available LED devices are generally not able to deliver multiple wavelengths or intensities, resulting in investigators employing multiple LED devices from different manufacturers, which may vary not only in the intensity, but also the spectrum of wavelength of light emitted. We have addressed this issue by employing a broadband Xenon light source filtered through narrowband interference filters, thereby generating a range of wavelengths and fluences of light, allowing close, accurate control of the parameters of R/NIR-LT.
It is important to note that the therapeutic dose of treatment is defined by the number of photons interacting with the photoacceptor (chromophore), which, in the case of R/NIR-LT is postulated to be cytochrome c oxidase (COX) 18. Photon energy delivered varies with wavelength; meaning equal doses of energy at different wavelengths will be comprised of different numbers of photons. Therefore, the light emitted from the device was calibrated to emit an equal number of photons for each of the chosen wavelengths to be tested. We have developed a system that can be used to deliver R/NIR-LT at a range of wavelengths and intensities to cells in vitro and demonstrated the ability to measure the effects of the delivered R/NIR-LT on ROS production in cells subjected to glutamate stress.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>OxiSelect Intracellular ROS Assay Kit (Green Fluorescence)</td>
			<td>Cell Biolabs</td>
			<td>STA-342</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex UltraRed Reagent</td>
			<td>Molecular Probes</td>
			<td>A36006</td>
			<td></td>
		</tr>
		<tr>
			<td>300 Watt Xenon Arc Lamp</td>
			<td>Newport Corporation</td>
			<td>6258</td>
			<td>Very intense light source, do not look directly into the lamp. Ensure there is sufficient cooling to the lamp whilst it is switched on</td>
		</tr>
		<tr>
			<td>USB4000-FL Fluorescence Spectrometer</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>CC-3-UV Cosine Corrector for Emission Collection</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>200&#956;m diameter quartz fibre optic</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraSuite Spectroscopy Platform</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>2300 EnSpire Multimode Plate Reader</td>
			<td>Perkin Elmer</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>9002-93-1</td>
			<td>Acute toxicity, wear gloves when handling.</td>
		</tr>
		<tr>
			<td>L-Glutamic acid monosodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>142-47-2 (anhydrous)</td>
			<td></td>
		</tr>
		<tr>
			<td>Pheochromocytoma rat adrenal medulla (PC12) cells</td>
			<td>American Type Culture Collection</td>
			<td>CRL-2522</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI1640) Media</td>
			<td>Gibco</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
			<td>Gibco</td>
			<td>10082-147</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Horse Serum, New Zealand origin</td>
			<td>Gibco</td>
			<td>16050-122</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>100 mM Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>100X MEM Non-Essential Amino Acids Solution</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Retinal Muller (rMC1) cells</td>
			<td>University of California, San Diego</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-118</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>75cm2 Flasks</td>
			<td>BD Biosciences</td>
			<td>B4-BE-353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>25988-63-0</td>
			<td>Aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14025-134</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-049</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein, Natural</td>
			<td>Gibco</td>
			<td>23017-015</td>
			<td>Aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>1X Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>165U Papain</td>
			<td>Worthington</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>W326305</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 96 well plates, clear bottom, black</td>
			<td>Corning</td>
			<td>CLS3603-48EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar Clear Polystyrene 96-Well Plates Untreated; Well shape: Round; Sterile.</td>
			<td>Costar</td>
			<td>07-200-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Seesaw Rocker</td>
			<td>Standard lab epuipment</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Standard lab epuipment</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral Density Filter Paper (0.3)</td>
			<td>THORLABS</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>442nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FL441.6-10</td>
			<td></td>
		</tr>
		<tr>
			<td>550nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB550-10</td>
			<td></td>
		</tr>
		<tr>
			<td>670nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB670-10</td>
			<td></td>
		</tr>
		<tr>
			<td>810nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB810-10e</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.1)</td>
			<td>THORLABS</td>
			<td>NE01B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.2)</td>
			<td>THORLABS</td>
			<td>NE02B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.3)</td>
			<td>THORLABS</td>
			<td>NE03B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.5)</td>
			<td>THORLABS</td>
			<td>NE05B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.6)</td>
			<td>THORLABS</td>
			<td>NE06B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (1.0)</td>
			<td>THORLABS</td>
			<td>NE10B</td>
			<td></td>
		</tr>
	</tbody>
</table>

method for the assessment of effects of a range of wavelengths and intensities of red/near-infrared light therapy on oxidative stress in vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

The output of light delivered at a wavelength of 670nm was calibrated using neutral density filters in order to irradiate cells with a range of fluences encompassing a dose of 670nm light previously shown to be beneficial in vivo (0.3 J/cm2) 20. As the number of neutral density filters in front of the light source increased, the intensity (W/m2) decreased, allowing less light to pass to the target area. Table 1 presents the calibration data of 670nm light generated from the light...

Watch this Scientific Journal Video about Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro at JoVE.com

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Research

JoVE Journal

Biology

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

Biochemical Titration of Glycogen In vitro

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...