Giacci Marcus K, Hart Nathan S, Hartz Richard V, Harvey Alan R, Hodgetts Stuart I, Fitzgerald Melinda
Experimental and Regenerative Neurosciences, School of Animal Biology, The University of Western Australia.
School of Animal Biology and The Oceans Institute, The University of Western Australia.
J Vis Exp. 2015 Mar 21(97):52221. doi: 10.3791/52221.
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 810 nm) and fluences (8.5x10(-3) to 3.8x10(-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.
通过激光或发光二极管（LED）进行的红/近红外光疗法（R/NIR-LT）可改善一系列中枢神经系统损伤在体内的功能和形态学结果，可能是通过减少氧化应激实现的。然而，已表明R/NIR-LT对氧化应激的影响会因照射波长或强度的不同而有所变化。由于缺乏可提供多种波长或强度、适用于高通量体外优化研究的商用设备，比较治疗参数的研究尚缺。本方案描述了一种在一系列波长和强度下输送光的技术，以优化给定损伤模型所需的治疗剂量。我们假设，一种能够轻松改变波长和强度参数的光输送方法，有助于确定R/NIR-LT在体外减少活性氧（ROS）的最佳剂量。非相干氙光通过窄带干涉滤光片进行过滤，以向培养细胞输送不同波长（中心波长为440、550、670和810 nm）和光通量（8.5×10⁻³至3.8×10⁻¹ J/cm²）的光。该装置的光输出经过校准，以便在每个波长下发射具有治疗相关性的等量量子剂量的光。使用DCFH-DA和对H₂O₂敏感的荧光染料，在经光处理的谷氨酸应激细胞中检测活性物质。我们成功地将一系列生理和治疗相关波长及强度的光输送到了作为中枢神经系统损伤模型暴露于谷氨酸的培养细胞中。虽然本研究中使用的R/NIR-LT光通量未对培养细胞产生的ROS产生影响，但光输送方法适用于其他系统，包括分离的线粒体或更具生理相关性的器官型切片培养模型，并且可用于评估对氧化代谢一系列结果指标的影响。
