金纳米棒辅助神经细胞的刺激光
Summary
This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.
Abstract
Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.
Introduction
最近的研究表明，用红外光的由水（波长> 1400纳米）的吸收相关的瞬态加热可以用于诱导动作电位的神经组织1，并在心肌2细胞内钙瞬变的。使用红外光的提出用于神经假体的应用极大的兴趣，由于潜在的更精细的空间分辨率，缺乏与组织去除的必要基因的直接接触，最小化刺激伪影，并修改之前刺激细胞（根据需要在光遗传学）1。尽管所有的这些优点，最近开发的热模型表明，目标组织/细胞可能受到的累积热效应，当多个刺激位点和/或高重复率使用3,4。
为了应对这些挑战，研究人员已经认识到使用外在absor电位的BER为神经刺激，以产生在组织中更局部加热效应。 Huang 等人通过使用超顺磁性铁氧体纳米远程激活温度敏感的TRPV1通道的HEK 293细胞与射频磁场表现出5这一原则。尽管这种技术可以允许更深的渗透（磁场相互作用相对较弱与组织），则反应进行仅记录在几秒钟期间，而不是在仿生器件5所需的毫秒的持续时间。大鼠皮质神经元用黑色微粒在体外的类似的，法拉等人证明电刺激。它们表现出刺激细胞级精度使用脉冲持续时间数百微秒和能量在μJ的范围的顺序，潜在地允许更快的重复率6。
使用外在吸收剂也被应用以诱导形态学变化的抑制作用 。 Ciofani 等显示在神经元细胞生长增加了〜40％的使用由超声7激发压电氮化硼纳米管。同样地，内吞氧化铁纳米颗粒在PC12细胞中已经报道了以提高神经突分化以剂量依赖的方式，由于细胞粘附分子的活化与氧化铁8。
近日，在吸收外在的利益，协助神经刺激还注重利用金纳米粒子（金纳米粒子）。金纳米粒子具有有效地吸收激光光线的电浆峰和消散它到周围环境中的热量9的形式的能力。在所有可用的粒子形状，金纳米棒的光吸收（凹NRS）方便地匹配的生物组织的治疗窗（近红外-红外，750-1,400纳米之间的波长）10。此外，在续神经刺激的电话分机，采用的Au卢比的提供了相对良好的生物相容性和广泛的表面功能选项11。最近的研究已经表明，在分化的刺激作用能够的Au卢比的NG108-15神经细胞12连续激光照射后所诱导。同样，细胞内钙瞬变记录后激光照射调制可变频率和脉冲长度13金自然保护区培养神经细胞。金自然保护区的螺旋神经节神经元14的原代培养NIR激光照射后，还记录了细胞膜去极化。首先在与辐照金自然保护区体内应用已被证明只是最近。 EOM和同事露出的Au卢比在其电浆峰和记录在6倍的增加的化合物的神经动作电位（CNAPS）的幅度和一个3倍的降低，在刺激阈值中的大鼠坐骨神经。带连接hanced响应归因于从NR电浆峰15的激发导致局部加热效应。
在本论文，调查激光刺激与金自然保护区培养NG108-15神经细胞的影响，协议规定。这些方法提供了一个简单，但强大的方式使用标准的生物技术和材料照射细胞群在体外 。所述协议是基于一个光纤耦合激光二极管（LD），允许安全操作和重复的排列。在Au NR样品制备和激光照射方法可以进一步扩展到不同的颗粒形状和神经元细胞培养物，提供了具体的合成和培养协议是已知的，分别。
Protocol
Representative Results
Discussion
在这个演讲中概述的协议描述了如何培养，区分和使用光学吸收外在刺激神经细胞。了NR特性（ 例如尺寸，形状，等离子体共振波长和表面化学）和激光的刺激参数（如波长，脉冲长度，重复率等 ）可以变化，以满足不同的实验的需要。可以使用标准的生物测定法和材料来监测对细胞行为的影响。总的来说，方法提供了一种简单，但功能强大，这样照射细胞群在体外 ，并且?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
笔者要感谢NanoVentures澳大利亚旅行的资金支持，并教授约翰·海科克为被部分托管于该研究在谢菲尔德和Jaimee梅恩女士的大学为她拍摄时的帮助。
Materials
| Au NR | Sigma Aldrich | 716812 | |
| NG108-15 | Sigma Aldrich | 8811230 | |
| DMEM | Sigma Aldrich | D6546 | |
| FCS | Life Technologies | 10100147 | |
| L-glutamine | Sigma Aldrich | G7513 | |
| Penicillin/streptomycin | Life Technologies | 15140122 | |
| Amphotericin B | Life Technologies | 15290018 | |
| Formaldehyde | Sigma Aldrich | F8775 | |
| Triton X-100 | BDH | T8532 | |
| BSA | Sigma Aldrich | A2058 | |
| Anti-βIII-tubulin | Promega | G7121 | |
| TRITC-conjugated anti-mouse IgG antibody | Sigma Aldrich | T5393 | |
| DAPI | Invitrogen | D1306 | |
| Fluo-4 AM | Invitrogen | F14201 | |
| DMSO | Sigma Aldrich | 472301 | |
| Pluronic F-127 | Invitrogen | P6867 | |
| Equipment name | Company | Catalogue Number | |
| UV-Vis spectrometer | Varian Medical Systems Inc. | Cary 50 Bio | |
| Mini centrifuge | Eppendorf | Mini Spin | |
| Sonic bath | Unisonics Australia | FPX 10D | |
| Cell culture incubator | Kendro | Hera Cell 150 | |
| Cell culture centrifuge | Hettich | Rotofix 32A | |
| Laser diode | Optotech | 780 nm single mode fibre – coupled LD | |
| Optical fiber | Thorlabs | 780 HP | |
| Power meter | Coherent | Laser Check | |
| ImageJ | http://rsb.info.nih.gov/ij/index.html | ||
| Epifluorescent microscope | Axon Instruments | ImageX-press 5000A | |
| μ-slide well | Ibidi | 80826 | |
| Inverted confocal microscope | Carl Zeiss Microscopy Ltd. | LSM 510 meta-confocal microscope | |
| Oscilloscope | Tektronix | TDS210 |
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