表征使用原子力显微镜，冲击压痕和流变测定脑组织的多尺度力学性能
1Department of Materials Science and Engineering,Massachusetts Institute of Technology, 2Department of Biological Engineering,Massachusetts Institute of Technology, 3Department of Mechanical Engineering,Massachusetts Institute of Technology, 4Department of Neurology, The F.M. Kirby Neurobiology Center,Boston Children’s Hospital, Harvard Medical School
Summary
We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.
Abstract
设计和工程师由大脑的属性激发的材料，无论是机械模拟物或组织再生的研究中，脑组织本身必须很好地表征在不同的长度和时间尺度。像许多生物组织，脑组织呈现出一个复杂的，分层结构。然而，与大多数其它组织，脑是非常低的机械刚度，以Pa的100s的顺序上杨氏弹性模量即这种低刚度可以呈现挑战关键的机械性能的实验表征。在这里，我们表明，已适应测量水分，符合生物材料的脑组织等，在不同的尺度和负荷率弹性和粘弹性能几种机械表征技术。在微尺度，我们进行使用原子力显微镜启用压痕蠕变遵守和力量松弛实验。在mesos凯尔，我们执行使用的是基于钟摆仪器化压压痕冲击实验。在宏观尺度，我们进行平行板流变量化该频率依赖剪切弹性模量。我们还讨论了与每个方法相关的挑战和限制。这些技术共同使脑组织，可用于更好地理解脑的结构和工程师仿生材料进行了深入的机械特性。
Introduction
包含生物器官最软组织是在机械上和结构复杂，低刚度相比矿化骨或工程材料，并显示出非线性的和取决于时间的变形。相比，在身体的其他组织，脑组织是非常柔顺的，以帕1的100秒的数量级上的弹性模量例如脑组织呈现出具有鲜明和相互交叉的灰质和白质区域也有所不同功能结构的异质性。理解脑组织力学会在材料和计算模型的设计有助于向模拟大脑损伤时的反应，促进机械损伤的预测，并启用的保护策略工程。此外，这样的信息可以用来考虑的组织再生的设计目标，以更好地理解脑组织是与疾病，如多发性硬化症和孤独症有关的结构变化。 HERE，我们描述和证明了可用来表征机械柔性组织中，包括脑组织的粘弹性能几个实验方法，在微观，中观和宏观尺度。
在微尺度，我们进行了蠕变遵守和用原子力显微镜（AFM） – 启用压痕力松弛实验。通常情况下，使能AFM压痕用于估计样品2-4的弹性模量（或瞬时刚度）。然而，同样的仪器也可用于测量微尺度粘弹性（时间或速率依赖性）性质：5-10。这些实验中，在图1所示的原理，是缩进悬臂探针进入脑组织的AFM，保持力或压痕深度指定大小，并测量在分别压痕深度和力，相应的变化，随着时间的推移。通过这些数据，我们可以计算出蠕变补偿分别lianceĴC和松弛模量G R，。
在尺度，我们在维持组织结构和水化水平液体浸泡条件下进行冲击试验压痕，使用的是基于钟摆仪器化纳米压痕。实验装置如图2所示，随着钟摆成与组织接触，探针直到振荡摆锤涉及到组织内休息位移记录为时间的函数。从探头所得阻尼谐振荡运动，我们可以计算出最大穿透深×max时 ，耗能能力K和耗散品质因数Q（其涉及能量耗散率）的组织11,12。
在宏观尺度，我们使用了平行板流变仪来量化该频率依赖剪切弹性模量，称为储能模量G'和损耗模量G“时，组织的。在这种类型的流变的，我们应用一个谐波角株（和对应的剪应变）在已知振幅和频率，并测量reactional扭矩（和相应的剪切应力） ， 如图3，从所测量的转矩的产生幅度和相位滞后和系统的几何变量，我们可以在感兴趣13,14施加的频率计算G'和G“。
Protocol
伦理学声明：所有的实验方案是由波士顿儿童医院的动物研究委员会批准并符合健康指南实验动物的护理和使用全国学院。 1.鼠标脑组织采集程序（启用了AFM压痕和影响压痕） 准备氯胺酮/甲苯噻嗪混合物麻醉小鼠。结合5毫升氯胺酮（500毫克/毫升），加入1ml甲苯噻嗪（20毫克/毫升）和7毫升0.9％盐溶液中。 每氯胺酮/甲苯噻嗪溶液克体重7微升注入小鼠?…
Representative Results
图4示出代表压痕和力与时间的反应（ 图4B，C）用于蠕变柔和力松弛实验中，给定分别施加的力或压痕深度（ 图4A中的D）。利用这些数据和系统的几何形状，蠕变柔量J C（t）和强制松弛模量ģR（T）可被计算为大脑（ 图4C中的F）的不同的区域。虽然以前的研究表明大脑23的不同区域?…
Discussion
本文提出的每个技术测量脑组织的机械性能的不同方面。蠕变柔和应力松弛模量随时间变化的机械性能的量度。储存和损耗模数代表率相关的机械性能。冲击压痕还测量速率依赖性机械特性，但在能量耗散的情况下。当表征组织的机械性能，同时启用AFM压痕和流变的常用方法。因为除了提供时间相关的材料性质，不同的实验参数可以用来测量细胞和组织的弹性模量4，甚至频率相关特性<sup…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We acknowledge support of this work by the National Multiple Sclerosis Society and Simons Center for the Social Brain. BQ acknowledges support from the U.S. National Defense Science & Engineering Graduate Fellowship program.
Materials
| Xylaxine | Lloyd Laboratoried | perscription drug | |
| Ketamine | AnaSed Injections | perscription drug | |
| Vibratome (Vibrating blade microtome) | Leica | VT1200 | |
| Hibernate-A Medium | Gibco | A1247501 | CO2-independent neural medium for adult tissue |
| Atomic Force Microscope, MFP-3D-BIO | Asylum Research | – | |
| Petri Dish Heater | Asylum Research | – | |
| AFM Probe, 0.03 N/m, 10 um radius borosilicate sphere | Novascan | PT.GS | |
| Cell-Tak | Corning | 354240 | mussel-derived bioadhesive |
| Sodium Bicarbonate | Sigma-Aldrich | S5761 | alternate suppliers can be used |
| Sodium Hydroxide, 1N | Sigma-Aldrich | 59223C | alternate suppliers can be used |
| Instrumented Indenter, NanoTest Vantage | Micro Materials Ltd. | – | probe tip needs to be machined (steel flat punch, 1mm diameter, 4-5 mm length) |
| NanoTest Liquid Cell | Micro Materials Ltd. | – | |
| Parallel Plate Rheometer MCR501 | Anton-Parr | – | |
| PP25 | Anton-Parr | – | 25 mm diameter flat measurement plate |
| Adhesive Sandpaper | McMaster-Carr | 4184A48 | alternate suppliers can be used |
| Loctite 4013 Instant Adhesive | Henkel | 20268 | alternate suppliers can be used |
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Citazione di questo articolo
Canovic, E. P., Qing, B., Mijailovic, A. S., Jagielska, A., Whitfield, M. J., Kelly, E., Turner, D., Sahin, M., Van Vliet, K. J. Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry. J. Vis. Exp. (115), e54201, doi:10.3791/54201 (2016).