对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em>在体外</em
Summary
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.
Abstract
红/近红外光疗法（R / NIR-LT），通过激光或发光二极管（LED）的递送，可能通过减少氧化应激改善功能和形态学效果的范围内的中枢神经系统损伤的体内 ，的。然而，在氧化应激的R / NIR-LT的作用已被证明取决于波长或辐射的强度而变化。在比较治疗参数的研究缺乏，由于缺乏能够提供多个波长或强度，适用于通过量高的体外优化研究商用设备。这个协议描述了用于递送光的技术在一定范围的波长和强度，以优化所需的一个给定的损伤模型治疗剂量。我们假设，提供光的方法，其中波长和强度参数可以很容易地被改变，可以促进确定的R / NIR-LT的最佳剂量的减少活性氧（ROS）的体外。
非相干氙光通过窄带干涉滤光片过滤，以提供不同的波长（440，550，670的中心波长和810纳米）和能量密度（8.5×10 -3至3.8×10 -1焦耳/ cm 2）的光的向培养的细胞中。从该装置的光输出被校准以发射治疗相关，等于量子剂量的光的每个波长。检测反应性物质在谷氨酸强调与光处理的细胞，用DCFH-DA和H 2 O 2的敏感的荧光染料。
我们成功地在一个范围内的生理学和治疗相关的波长和强度的光传递，以暴露于谷氨酸作为CNS损伤模型的培养细胞。而R / NIR-LT在当前研究中使用没有施加由培养的细胞产生的活性氧的效果的能量密度，轻的递送方法也适用于其他SYSTEMS包括分离线粒体或多种生理上相关的器官切片培养模型，并且可以用来评估的范围内的氧化代谢的结果的措施的效果。
Introduction
活性氧（ROS）的所需要的各种信号转导途径和细胞代谢的正常反应，包括那些神经保护1。然而，当内源性抗氧化机制无法控制ROS的产生，细胞可能会屈从于氧化应激2,3。损伤后的中枢神经系统，在ROS和氧化应激的存在相关联的增加被认为在损伤4,5的进展显着的作用。尽管用于衰减已评估氧 化应激策略的广泛的数目，目前有用于以下神经外伤6衰减ROS产生和相关的氧化应激在临床使用中没有完全有效，临床相关抗氧化剂的策略。因此氧化应激的衰减仍是治疗介入7的一个重要目标。
后续的改进荷兰国际集团的R / NIR-LT已经报道了在广泛的伤害和疾病，包括减少心肌梗死面积，糖尿病期间肾和肝的并发症，视网膜变性，中枢神经系统损伤和中风8，或许通过减少氧化应激。特别是关于CNS损伤，670nm处光的功效的临床前研究已经显示在视网膜变性9-11，脊髓损伤12，神经元死亡13的模型良好的效果。临床试验已经进行了干性年龄相关性黄斑变性和目前正在进行对于中风14，但是这些试验的结果不出现希望的，或许是由于未能采用有效的治疗参数15。因此，R / NIR-LT并没有被广泛采用作为在神经外伤正常临床实践的一部分，尽管是一种简单的施用，非侵入性的和相对便宜的治疗方法。障碍的临床翻译，包括缺乏CL的早知道行动规范有效的治疗方案16,17和缺乏机制。关于光疗法目前的文献揭示了过多的变化治疗参数相对于照射源（LED或激光），波长（ 例如 ，630，670，780，810，830，880，904nm），照射总剂量（焦耳/单位面积），持续时间（曝光时间），定时（前或后损伤），治疗的频率和传递模式（脉冲或连续）8。在研究之间的治疗参数的变化，使比较困难，已经向有关的功效持怀疑态度16贡献。
因此，R / NIR-LT的优化显然需要，与细胞培养系统能够提供必要的比较的多个变量的高通量筛选的机制。然而也有一些市售的照明系统，可以提供足够的灵活性和控制わvelength和强度进行这样的优化实验。市售的LED器件通常是不能够提供多个波长或强度，从而导致研究者使用来自不同制造商，其可以不仅在强度变化的多个LED器件，而且光发射的波长的光谱。我们已通过使用宽带氙光源透过窄带干涉滤光片过滤，从而产生范围内的波长和光的能量密度，从而允许R / NIR-LT的参数密切，精确控制解决了这一问题。
要注意的是治疗的治疗剂量由光子与photoacceptor（发色团），其中在R / NIR-LT的情况下被假定为细胞色素C氧化酶（COX）18相互作用的数目定义是很重要的。输送光子能量随波长;意相等剂量的能量以不同的波长将是玉米珍贵的不同数量的光子的。因此，从装置发射的光被校准以发射相等数目的光子的每个所选择的波长进行测试。我们已开发了可用于递送的R / NIR-LT在一定范围的波长和强度，以细胞在体外的系统和演示来测量递送的R /对ROS产生NIR-LT在细胞经受影响的能力谷氨酸压力。
Protocol
Representative Results
Discussion
我们已经成功地适应了精确和校准的光传输系统，以提供一种机制，用于对R / NIR-LT 在体外优化的研究。的R波长和强度的参数/ NIR-LT能够被准确有效地操纵使用这个系统。我们建立了光治疗的细胞并没有导致细胞死亡，尽管ROS没有减少在输送的波长和剂量，在测试的细胞类型。当前系统在670nm处（20.11W /米2）实现的最大强度在过量照射的外显率的先前公布的措施到大鼠脑，其中1.17瓦…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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 |
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