使用石墨烯电极阵列进行脑映射
Summary
我们提出了一种基于石墨烯阵列的脑映射程序，以减少侵入性并提高时空分辨率。基于石墨烯阵列的表面电极表现出长期的生物相容性、机械灵活性和在卷曲大脑中用于大脑映射的适用性。该协议允许同时和顺序构建多种形式的感官图谱。
Abstract
皮质图表示大脑皮层中对感觉运动刺激的位置依赖性神经反应的空间组织，从而能够预测生理相关行为。各种方法，如穿透电极、脑电图、正电子发射断层扫描、脑磁图和功能性磁共振成像，已被用于获得皮质图。然而，这些方法受到时空分辨率差、信噪比（SNR）低、成本高、非生物相容性或对大脑造成物理损害的限制。本研究提出了一种基于石墨烯阵列的体感映射方法，作为皮质电图的一个特点，该方法具有优异的生物相容性、高时空分辨率、理想的信噪比和最小的组织损伤，克服了以前方法的缺点。本研究证明了石墨烯电极阵列在大鼠体感映射中的可行性。所提出的方案不仅可以应用于躯体感觉皮层，还可以应用于其他皮层，例如听觉、视觉和运动皮层，为临床实施提供先进的技术。
Introduction
皮质图是一组局部斑块，表示大脑皮层中对感觉运动刺激的反应属性。它们是神经网络的空间形成，能够预测感知和认知。因此，皮质图可用于评估对外部刺激的神经反应和处理感觉运动信息1,2,3,4。有创和非侵入性方法可用于皮质映射。最常见的侵入性方法之一是使用皮质内（或穿透）电极来映射5,6,7,8。
使用穿透电极评估按需高分辨率皮质图面临着几个障碍。该方法太费力，无法获得像样的地图，而且侵入性太强，无法在临床上使用，从而阻碍了进一步发展。脑电图 （EEG）、正电子发射断层扫描 （PET）、脑磁图 （MEG） 和功能性磁共振成像 （fMRI） 等最新技术越来越受欢迎，因为这些技术侵入性较小且可重复。然而，鉴于其高昂的成本和解决率低，它们在有限数量的案例中使用9,10,11。近年来，具有优异信号可靠性的柔性表面电极引起了广泛的关注。基于石墨烯的表面电极表现出长期的生物相容性和机械灵活性，在复杂的大脑中提供稳定的记录12,13,14,15,16。我们小组最近开发了一种基于石墨烯的多通道阵列，用于皮质表面的高分辨率记录和位点特异性神经刺激。这项技术使我们能够长时间跟踪感官信息的皮层表征。
本文介绍了使用 30 通道石墨烯多电极阵列获取体感皮层大脑图所涉及的步骤。为了测量大脑活动，将石墨烯电极阵列放置在皮层的硬膜下区域，同时用木棍刺激前爪，前肢，后爪，后肢，躯干和胡须。记录体感区域的体感诱发电位 （SEP）。该协议也可以应用于其他大脑区域，例如听觉，视觉和运动皮层。
Protocol
所有动物处理程序均已获得仁川国立大学机构动物护理和使用委员会的批准（INU-ANIM-2017-08）。 1. 动物手术准备 注意：使用Sprague Dawley Rat（8-10周大）进行本实验，没有性别偏见。 腹膜内用90mg / kg氯胺酮和10mg / kg甲苯噻嗪混合物麻醉大鼠。为了在整个手术过程中保持所需的麻醉深度，当大鼠出现醒来迹象时，提供补充 45 mg/kg 氯胺酮和 5…
Representative Results
该协议描述了石墨烯多通道阵列如何安装在大脑表面。通过获取对物理刺激的神经反应并计算反应的幅度来构建体感图。 图1 显示了该实验的示意图。 图2A 显示了石墨烯电极阵列的结构特征。电极之间有基板的通孔。这些孔有助于电极牢固地接触皮质表面（图2B）。电极与皮层的强粘附有助于以更少的噪音记…
Discussion
所提出的协议提供了一个深入的分步过程，解释了如何使用石墨烯电极阵列访问和映射大鼠的体感反应。协议获取的数据是标准必要专利，提供与每个身体部位突触相关的躯体感觉信息。
应考虑该协议的几个方面。在提取脑脊液以防止脑水肿和减轻炎症时，实验者不要损坏位于大池前面的脑干至关重要。
面部胡须提供有关周围环境的触觉感官信息，例如…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了仁川国立大学（国际合作）对Sunggu Yang的支持。
Materials
| 1mL syringe | KOREAVACCINE CORPORATION | injecting the drug for anesthesia | |
| 3mL syringe | KOREAVACCINE CORPORATION | injecting the drug for anesthesia | |
| Bone rongeur | Fine Science Tools | 16220-14 | remove the skull |
| connector | Gbrain | Connect graphene electrode to headstage | |
| drill | FALCON tool | grind the skull | |
| drill bits | Osstem implant | grind the skull | |
| Graefe iris forceps slightly curved serrated | vubu | vudu-02-73010 | remove the tissue from the skull or hold wiper |
| graphene multielectrode array | Gbrain | records signals from neuron | |
| isoflurane | Hana Pharm Corporation | sacrifce the subject | |
| ketamine | yuhan corporation | used for anesthesia | |
| lidocaine(2%) | Daihan pharmaceutical | local anesthetic | |
| Matlab R2021b | Mathworks | Data analysis Software | |
| mosquito hemostats | Fine Science Tools | 91309-12 | fasten the scalp |
| ointment | Alcon | prevent eye from drying out | |
| povidone | Green Pharmaceutical corporation | disinfect the incision area | |
| RHS 32ch Stim/Record headstage | intan technologies | M4032 | connect connector to interface cable and contain intan RHS stim/amplifier chip |
| RHS 6-ft (1.8m) Stim SPI interface cable | intan technologies | M3206 | connect graphene electrode to headstage |
| RHS Stim/Recording controller software | intan technologies | Data Acquisition Software | |
| RHS stimulation/ Recording controller | intan technologies | M4200 | |
| saline | JW Pharmaceutical | ||
| scalpel | Hammacher | HSB 805-03 | |
| stereotaxic instrument | stoelting | fasten the subject | |
| sterile Hypodermic Needle | KOREAVACCINE CORPORATION | remove the dura mater | |
| Steven Iris Tissue Forceps | KASCO | 50-2026 | remove the dura mater |
| surgical blade no.11 | FEATHER | inscise the scalp | |
| surgical sicssors | Fine Science Tools | 14090-09 | inscise the scalp and remove the dura mater |
| wooden stick | whisker stimulation | ||
| xylazine | Bayer Korea | used for anesthesia |
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Citer Cet Article
Kim, D., Jeong, M., Kim, E., Kim, G., Na, J., Yang, S. Brain Mapping Using a Graphene Electrode Array. J. Vis. Exp. (200), e64910, doi:10.3791/64910 (2023).