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.
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.
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.
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.
1. Optical Calibration: Measuring Light Output
Figure 1. Image of the light delivery apparatus. Illustrated are the light power source, xenon lamp with housing, collimating lens, water filter, entrance aperture, liquid light guide, second collimating lens, filter holder, treatment frame and matte black card. Note that the narrowband wavelength and intensity filters are not shown.
2. Cell Preparation
3. Adding Glutamate Stressor to Cells
4. First Dosages of Light Treatment
5. Final Dosage of Light Treatment and Detection of ROS
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 source fitted with a wavelength filter and includes the number of ND filters used and the intensity of light generated as a result, at described distances from the light output. Fluence, or dose of 670nm light (J/cm2), was calculated from the equation: [Dose (J/cm2) = (Light intensity (W/m2) / 10,000) x time (s)], where the time of treatment was 180s.
Number of ND filters | Distance from light output (cm) | Intensity (W/m^2) | Dose (J/cm^2) |
0 | 10.5 | 20.11 | 0.38 |
0 | 14 | 10.55 | 0.19 |
1 | 14 | 4.91 | 0.075 |
2 | 14 | 2.28 | 0.041 |
3 | 14 | 1.03 | 0.018 |
4 | 14 | 0.47 | 0.0085 |
5 | 14 | 0.21 | 0.0038 |
6 | 14 | 0.094 | 0.00169 |
7 | 14 | 0.045 | 0.00081 |
8 | 14 | 0.021 | 0.000378 |
9 | 14 | 0.013 | 0.000234 |
Table 1: Output of the light delivery apparatus fitted with the 670nm wavelength filter.Number of ND filters refers to the number of neutral density filters fitted to the front of the light source output. Intensity (W/m2) refers to the intensity of the light as reported by the propriety software. Fluence, or dose was calculated by the equation[Dose (J/cm2) = (Light intensity (W/m2) / 10,000) x time (s)], where the time of exposure was 180s and distance from the light output was 10.5 or 14 cm.
Two clinically relevant quantal fluences of light were chosen to investigate differential effects of R/NIR-LT wavelength on production of ROS. A dose that can reach CNS tracts following transmission through overlying tissue using LED devices(i.e., 1.78 W/m2 at 670nm) 20 equated to 0.03 J/cm2 for a 3 min treatment, or 4.9 x 1014 photons/cm2/s. The light source equipped with filters to result in emission of 442, 550, 670 or 830nm was then calibrated using combinations of neutral density filters to emit equal quantal outputs (photons) for each wavelength as opposed to energy outputs (J/cm2), and the dosages (J/cm2) and intensities in W/m2 calculated (Table 2a). An additional higher dose that was within the recommended guidelines to stimulate cellular activity 21 was also used (1.29 x 1015 photons/cm2/s), and calibration conducted for each wavelength (Table 2b).
Wavelength (λ) | Dose (J/cm^2) | Intensity (W/m^2) | Emission (Photons/cm^2/s) |
442 | 0.057 | 3.21 | 4.8 x 10^14 |
550 | 0.051 | 2.87 | 5.0 x 10^14 |
670 | 0.032 | 1.78 | 4.9 x 10^14 |
830 | 0.018 | 1.01 | 4.9 x 10^14 |
Table 2: Calibration of intensity of dosage delivered by equal numbers of photons of light at varying wavelengths. Intensity (W/m2), dosage for a 3 min treatment (J/cm2) and emission (photons/cm2/s) when output of xenon light emitting 442, 550, 670 and 830nm are calibrated to emit A) 4.9 x 1014 photons/cm2/s or B) 1.3 x 1015 photons/cm2/s at a distance of 14 cm from the light output.
In order to assess whether the light delivered by the apparatus was toxic to cells at the dosages used, we assessed the protein content remaining in PC12 cell culture wells following the ROS assays, using a colorimetric protein assay. There was no significant loss of protein at any of the higher output dosages of the light (P > 0.05), indicating that the dosage of light delivered was not causing cell death (Figure 2) and is appropriate to use for assessments of oxidative metabolism.
Figure 2: Effect of varying doses of light on the total protein concentration remaining in culture wells following R/NIR-LT and ROS assay. Histogram bars are the mean ± S.E.M protein concentrations in PC12 cell culture wells, 6 replicates / concentration, experiments were repeated 3 times. There were no statistically significant differences between control and any of the treatment groups as determined by analysis of variance (ANOVA), p > 0.05.
We initially assessed the effects of 670nm light, delivered at fluences ranging from 0.0085 to 0.38 J/cm2, as an example wavelength to assess suitability of the light delivery apparatus. No significant effect of 670nm light was observed at any of the fluences tested when assessing either H2O2 or DCF fluorescence in PC12, rMC1 or mixed retinal cells stressed with glutamate (Figure 3, P > 0.05). Similarly, there were no significant effects of varying wavelengths of R/NIR-LT delivered at 4.9 x 1014 photons/cm2/s or 1.3 x 1015 photons/cm2/s on ROS production, when assessing H2O2 or DCF fluorescence (P > 0.05, data not shown). Our ability to detect changes in reactive species is confirmed by an increase in fluorescence of the DCFH-DA reactive dye at 13.44 ± 0.67 mM glutamate to 22.10 ±2.10 at 10mM glutamate. While our data do not reveal positive effects of R/NIR-LT delivered using our light delivery apparatus on ROS production in the selected model system, neither were there negative effects as cells were not compromised, indicated by sustained protein content in culture wells following light therapy (Figure 2). As such, the described method provides a protocol for treating cells or mitochondria with a defined dosage of photons at a range of wavelengths and may be used to assess higher dosages and alternative outcome measures, which may enable optimization of R/NIR-LT parameters.
Figure 3: Quantification of H2O2 (A-C) and DCF (D-F) fluorescence in PC12 (A, D), rMC1 (B, E) or mixed retinal cell (C, F) cultures in the presence of 10mM glutamate stressor, following 670nm irradiation therapy at fluence doses ranging from 0 – 0.38 J/cm2. Histogram bars represent the mean arbitrary fluorescence units / µg protein ± SEM. There were no statistically significant differences between control and any of the treatment groups as determined by ANOVA, p > 0.05, 6 replicates per group, experiments were repeated 3 times.
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/m2) are in excess of previously published measures of penetrance of irradiation into the rat brain, where 1.17 W/m2 reached the ventral surface of the optic nerve and 0.3W/m2 reached the ventral surface of the brain case. When delivered for 30 min, the dose received by cells of the optic nerve in vivo is therefore 0.054 – 0.31 J/cm2. The doses of 670nm light delivered in the current study (up to 0.38J/cm2) therefore encompass those received by cells in our previous in vivo studies.
The most widely accepted theory of R/NIR-LT efficacy is that of a photoacceptor based system 18, therefore it is imperative to ensure that cells treated with varying wavelengths receive equal quantal fluences of light (photons/cm2/s) 8. In addition, the exposure time used to deliver a particular fluence of light must be kept consistent between treatments, with only the intensity of the light changing to increase and decrease total fluence over time. Previous studies have demonstrated that changing the exposure time to deliver equal fluences results in differences in outcome measures and may act as a confounding factor, and hinder the identification of the optimal wavelength / fluence parameters required for a particular injury model 22,23. As such, we recommend the careful calibration of dosage at each wavelength tested, to ensure useful optimization of R/NIR-LT.
The described technique allows delivery of light to a range of in vitro model systems including organotypic slice cultures, which may result in more meaningful outcomes due to the preservation of cellular architecture and complex inter-cellular interactions. Variations on the injury model may require differences in treatment parameters of R/NIR-LT. This technique is limited by the maximum power output achievable with the described instrumentation. Higher power outputs may be required for some in vitro and most in vivo applications. If higher outputs of light are required, the apparatus may be altered to increase the intensity of light reaching the samples. Shortening the distance between the light termination and target cells will increase the intensity of light reaching the plate. Alternatively, the light may be reflected off a mirror and onto the cells as opposed to being filtered through a liquid light guide, however alternative wavelength and neutral density filters will be required to achieve this. Higher outputs using a more powerful light source would also be required if the method were to be adapted for delivery of R/NIR-LT in in vivo models of CNS injury, due to the increased dosages required to ensure effective penetrance of irradiation 8,20.
In conclusion, the current study describes a novel method of light delivery that provides a means of effectively altering intensity and wavelength parameters of light in R/NIR-LT studies. This methodology will be useful in optimization of R/NIR-LT employing a range of outcome measures and in vitro injury models.
The authors have nothing to disclose.
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.
OxiSelect Intracellular ROS Assay Kit (Green Fluorescence) | Cell Biolabs | STA-342 | |
Amplex UltraRed Reagent | Molecular Probes | A36006 | |
300 Watt Xenon Arc Lamp | Newport Corporation | 6258 | Very intense light source, do not look directly into the lamp. Ensure there is sufficient cooling to the lamp whilst it is switched on |
USB4000-FL Fluorescence Spectrometer | Ocean Optics | – | |
CC-3-UV Cosine Corrector for Emission Collection | Ocean Optics | – | |
200μm diameter quartz fibre optic | Ocean Optics | – | |
SpectraSuite Spectroscopy Platform | Ocean Optics | – | |
2300 EnSpire Multimode Plate Reader | Perkin Elmer | – | |
Pierce BCA Protein Assay Kit | Thermo Scientific | 23225 | |
Triton X-100 | Sigma-Aldrich | 9002-93-1 | Acute toxicity, wear gloves when handling. |
L-Glutamic acid monosodium salt hydrate | Sigma-Aldrich | 142-47-2 (anhydrous) | |
Pheochromocytoma rat adrenal medulla (PC12) cells | American Type Culture Collection | CRL-2522 | |
Roswell Park Memorial Institute (RPMI1640) Media | Gibco | 11875-119 | |
Fetal Bovine Serum, certified, heat inactivated, US origin | Gibco | 10082-147 | Warm to 37°C in water bath before use |
Horse Serum, New Zealand origin | Gibco | 16050-122 | Warm to 37°C in water bath before use |
GlutaMAX Supplement | Gibco | 35050-061 | Warm to 37°C in water bath before use |
100 mM Sodium Pyruvate | Gibco | 11360-070 | Warm to 37°C in water bath before use |
Penicillin-Streptomycin (10,000 U/mL) | Gibco | 15140-122 | Warm to 37°C in water bath before use |
100X MEM Non-Essential Amino Acids Solution | Gibco | 11140-050 | Warm to 37°C in water bath before use |
Retinal Muller (rMC1) cells | University of California, San Diego | – | |
Dulbecco's Modified Eagle Medium (DMEM) | Gibco | 11965-118 | Warm to 37°C in water bath before use |
75cm2 Flasks | BD Biosciences | B4-BE-353136 | |
Poly-L-lysine hydrobromide | Sigma-Aldrich | 25988-63-0 | Aliquot and store at -20°C |
Hank's Balanced Salt Solution (HBSS) | Gibco | 14025-134 | Warm to 37°C in water bath before use |
Phosphate-Buffered Saline (PBS) | Gibco | 10010-049 | Warm to 37°C in water bath before use |
Laminin Mouse Protein, Natural | Gibco | 23017-015 | Aliquot and store at -20°C |
1X Neurobasal Medium | Gibco | 21103-049 | Warm to 37°C in water bath before use |
Trypan Blue Solution, 0.4% | Gibco | 15250-061 | |
165U Papain | Worthington | – | |
L-Cysteine | Sigma-Aldrich | W326305 | |
Corning 96 well plates, clear bottom, black | Corning | CLS3603-48EA | |
Costar Clear Polystyrene 96-Well Plates Untreated; Well shape: Round; Sterile. | Costar | 07-200-103 | |
Seesaw Rocker | Standard lab epuipment | – | |
Centrifuge | Standard lab epuipment | – | |
Neutral Density Filter Paper (0.3) | THORLABS | – | |
442nm Bandpass Filter | THORLABS | FL441.6-10 | |
550nm Bandpass Filter | THORLABS | FB550-10 | |
670nm Bandpass Filter | THORLABS | FB670-10 | |
810nm Bandpass Filter | THORLABS | FB810-10e | |
Unmounted Ø25 mm Absorptive Neutral Density Filters (0.1) | THORLABS | NE01B | |
Unmounted Ø25 mm Absorptive Neutral Density Filters (0.2) | THORLABS | NE02B | |
Unmounted Ø25 mm Absorptive Neutral Density Filters (0.3) | THORLABS | NE03B | |
Unmounted Ø25 mm Absorptive Neutral Density Filters (0.5) | THORLABS | NE05B | |
Unmounted Ø25 mm Absorptive Neutral Density Filters (0.6) | THORLABS | NE06B | |
Unmounted Ø25 mm Absorptive Neutral Density Filters (1.0) | THORLABS | NE10B |