一种基于微 ct 的小动物脑损伤和定位电极表征方法
Summary
本文介绍了一种简单的方法来准备小动物大脑的微 ct 成像, 其中病变可以量化和电极定位在整个大脑的上下文中具有较高的精度。
Abstract
损伤和电极位置验证传统上是通过对染色脑片的组织学检查来完成的, 这是一个耗时的过程, 需要手动估计。在这里, 我们描述了一种简单、直接的方法, 用于量化大脑中的病变和定位电极, 这种方法不那么费力, 产生更详细的结果。整个大脑被雷亚铁污染, 嵌入树脂中, 并用微型 ct 扫描仪成像。扫描结果是大脑的3d 数字体积, 其分辨率和虚拟截面厚度取决于样本大小 (大鼠和斑马雀的每体素分别为12–15和 5-6μm)。表面和深层病变可以被定性, 单型或状物、四极体阵列、电解损伤和硅探针也可以局部化。自由和专有的软件允许实验者从任何平面上检查样品体积, 并手动或自动分割体积。由于这种方法产生整个大脑体积, 病变和电极的量化程度远远高于目前的方法, 这将有助于规范研究内部和研究之间的比较。
Introduction
神经学家长期依靠病变来了解大脑中的功能和位置之间的关系。例如, 我们对海马体是学习和记忆所不可缺少的理解, 对前额叶皮层作为冲动控制的关键的理解, 都是人类1,2的意外病变的产物。然而, 动物模型的使用, 使神经科学家能够通过超越偶然来利用病变的力量, 通过对结构-功能关系的系统研究, 已经阐明了无数大脑区域的功能。病变3,4。
然而, 要正确地将功能分配给结构, 病变研究需要精确的量化程序, 这是一个一直缺乏的领域。目前用于定量病变的金标准是用光学显微镜对大脑进行分割、安装和成像。然后将成像切片与地图集上最接近的部分进行匹配, 并间接报告受试者之间病变的大致坐标, 通常是通过使用相机 lucida 图像或组织切片3,4 示例 ,5,6,7,8,9, 10.
除了目前病灶定量程序的不精确, 这些技术是耗时和容易失败。大脑僵硬、刀片锐度和温度的微小变化会导致部分出现错误、扭曲或撕裂。由于安装介质中的气泡, 截面也会不均匀地染色和成像不当。重要的是, 在切片时, 病变在大脑中的位置的三维上下文丢失, 使大脑中病变的精确三维重建具有挑战性。
病变的另一个常见应用是确定大脑中单个和多个电极记录的位置。在最后的记录结束时, 研究人员在电极尖端诱导小的电解质病变, 并按照常规病变实验11的方式处理大脑的组织学.这种技术也有上述相同的缺点, 还有其他问题, 即电解质病变通常比用来制造电解质病变的电极大, 但通常足够小, 很难找到组织学上的东西。当插入多个电极时, 如四极阵列的情况下, 通过电解损伤进行验证更具挑战性。电解病变的另一种选择是在电极上使用染料来验证后的组织学 12, 但这种技术也有传统组织学的缺点。
在这里, 我们深入地描述了最近描述的方法13基于染色技术的电子显微镜 (em) 和 x 射线计算机断层扫描 (微 ct), 量化病变和定位电极的小动物大脑比目前更好方法。微 ct 是一种成像技术, 在这种技术中, x 射线拍摄于360°旋转的样品, 而闪烁器收集的 x 射线不会被样品偏转。其结果是对样品进行高分辨率的数字3d 重建, 可以在任何方向上进行可视化并精确量化。许多学术机构都有微型 ct 扫描仪, 这些扫描仪也可在商业上买到。
Protocol
Representative Results
Discussion
以下是协议的关键步骤: 首先, 使用 pfa 和 ga 的组合来灌注动物, 然后在修复后的大脑是至关重要的, 以实现对组织的一致的充分渗透。虽然我们没有明确地测试这一点, 一个合理的解释是, pfa 固定是可逆的 15, 而 ga 固定是不可逆的16,17。由于在四氧化铬中完全浸润组织需要两周的孵育, 因此大脑内部的 pfa 可能会扩散, 而在染色过程中组?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢格雷格·林和亚瑟·麦克莱伦在微 ct 机器方面的专业知识, 感谢哈佛医学院图像和数据分析核心 (idac) 的大卫·里士满和亨特·埃利奥特的图像处理建议, 以及波士顿的威廉·利伯蒂慷慨地提供斑马雀大脑的大学。这项工作部分是在国家纳米技术协调基础设施网络 (nnci) 的成员—-纳米渗透系统中心 (cns) 进行的, 该网络得到国家科学基金会第1541995年1541959国家科学基金会的支持。中枢神经系统是哈佛大学的一部分。这项工作得到了理查德和苏珊·史密斯家庭基金会以及 iarpa (合同 #D16PC00002) 的支持。s. b. e. w. 得到了人类前沿科学方案研究金的支持;lt000550 2014) 和欧洲分子生物学组织 (embo;altf1561-2013)。g. g. 得到了国家科学基金会 (nsf) 研究生研究奖学金计划 (grfp) 的支持。
Materials
| Paraformaldehyde (PFA) | Electron Microscopy Sciences (EMS) | 15710 | 2% (w/v/) in 1X PBS |
| Glutaraldehyde (GA) | EMS | 16220 | 2.5% (w/v) GA in 1X PBS |
| OsO4 | EMS | 19190 | Work in fume hood |
| Ethanol | Decon Labs | Koptec | 140, 190, 200 proof |
| Acetone | EMS | 10015 | Glass-distilled |
| Durcupan ACM resin | Sigma-Aldrich | 44610 | A, B, C and D components, resin for embedding |
| Disposable molds | Ted Pella | 27114 | Suggested |
| milliQ water (ultrapure water) | Millipore Sigma | QGARD00R1 (or related purifier) | Suggested |
| Parafilm (paraffin film) | Millipore Sigma | P7793 | Suggested paraffin film |
| Micro-CT scanner | Nikon Metrology Ltd., Tring, UK | X-Tek HMS ST 225 | Used by authors |
| Software for visualizing and analyzing micro-CT scans: | |||
| Volume Graphics | VG Studio Max | Used by authors | |
| FEI / Thermo Scientific | Avizo | Used by authors | |
| FEI / Thermo Scientific | Amira | Similar to Avizo | |
| Mark Sutton & Russell Garwood | Spiers | Free, http://spiers-software.org/ | |
| Pixmeo Sarl | Osirix Lite | Free, https://www.osirix-viewer.com/ | |
| Open Source | FIJI | Free, https://fiji.sc/ | |
| Adobe | Photoshop | Good for analyzing one slice at a time |
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