清醒小鼠海马毛细血管的体内纤维耦合临床前共聚焦激光扫描内窥镜 （pCLE）
Summary
虽然多光子成像仅在距组织表面的有限深度有效，但可以通过pCLE在任何深度实现3μm分辨率成像。在这里，我们提出了一种进行pCLE成像的方案，以测量发作期和野生型小鼠海马体中的微血管动力学。
Abstract
该协议的目的是描述光纤束耦合的临床前共聚焦激光扫描内窥镜（pCLE）的具体应用，以阐明癫痫发作期间由壁细胞驱动的毛细血管血流效应。体外和体内皮质成像表明，由周细胞驱动的毛细血管收缩可由健康动物的功能性局部神经活动以及药物应用引起。在这里，提出了如何使用pCLE确定微血管动力学在任何组织深度（特别是在海马体中）癫痫神经变性中的作用的方案。我们描述了一种头枕技术，该技术已被用于记录清醒动物的pCLE，以解决麻醉剂对神经活动的潜在副作用。使用这些方法，可以在大脑的深层神经结构中进行数小时的电生理和成像记录。
Introduction
与其他显微成像方法1,2,3,4,5,6,7,8相比，基于光纤的活体共聚焦显微镜允许以高速（高达240 Hz，取决于视场大小9）测量任何大脑区域、任何深度、任何深度的血流动力学).光纤探针能够以 3 μm 的分辨率进行体内共聚焦激光扫描成像，因为探针的尖端（由一束 5000-6000 根直径为 3 μm 的单根光纤组成的无透镜物镜）可以以微电极的精度定位在目标荧光靶标的 15 μm 范围内。与体内双光子成像一样，必须事先将荧光团引入成像靶标。例如，可以将荧光素葡聚糖（或量子点）注射到脉管系统中，或者可以将遗传编码的荧光蛋白转染到细胞中，或者在成像之前将荧光染料（如俄勒冈绿BAPTA-1）批量加载到细胞中。
最近使用这些技术的研究发现，导致发作性毛细血管痉挛的壁细胞运动活动（癫痫发作期间壁细胞位置突然收缩9）可导致发作性海马神经退行性变9。虽然先前的影像学研究显示体外和体内周细胞收缩与药物应用有关 6,7,10,11,12，但 Leal-Campanario 等人发现了小鼠大脑中体内自发毛细血管收缩的第一个证据。为了确定与人类颞叶癫痫的相关性，他们研究了雄性（P30-40 岁）敲除 （KO） Kv1.1 （kcna1-null） 小鼠 14,15 （JAX stock #003532），这是人类发作性共济失调 1 型的遗传模型15.周细胞在自发性癫痫动物及其野生型 （WT） 同窝动物中驱动病理性和生理性海马壁画血管收缩9。这些观察结果在用红藻氨酸使癫痫的WT动物中复制，从而表明它们适用于其他形式的癫痫。此外，Leal-Campanario等人使用新颖的立体显微镜方法确定，癫痫动物的凋亡神经元（但不是健康的神经元）在空间上与海马微血管系统耦合。由于兴奋性毒性与脉管系统没有已知的空间关联，因此该结果表明异常毛细血管痉挛性缺血引起的缺氧会导致癫痫的神经退行性变。图 1 显示了总体设置的原理图。
Protocol
Representative Results
Discussion
我们开发了一种头帽约束系统，用于在清醒小鼠中同时进行电生理和光纤 pCLE 实验，从而减少麻醉药物引起的潜在反应污染。头盖和安装装置结构简单，可重复用于慢性清醒成像实验。我们根据体内显微血流成像的金标准TPLSM检查了记录的质量。
熟练的手术技能是实施我们在这里描述的方案的必要条件。手术必须在无菌条件下进行，并始终在手术显微镜下进行，同时在海马体…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
该项目由美国癫痫学会的研究倡议奖、亚利桑那州生物医学研究委员会授予SLM的奖项以及预防失明研究公司向纽约州立大学下州健康科学大学眼科系提供的挑战资助，纽约州帝国创新计划， 以及美国国家科学基金会（0726113、0852636和1523614）、巴罗神经学基金会、玛丽安·罗谢尔夫人、格蕾丝·韦尔顿夫人和尊严健康 SEED 奖的进一步资助，以及美国国家科学基金会（0726113、0852636 和 1523614）和美国国立卫生研究院（R01EY031971 AND R01CA258021 奖项）对 SLM 和 SMC 的进一步资助。 这项工作也得到了负责卫生事务的助理国防部长办公室的支持，奖励编号为。W81XWH-15-1-0138，至 S.L.M. L.-C.得到了西班牙教育部José Castillejo奖学金的支持。我们感谢 O. Caballero 和 M. Ledo 提供的技术咨询和援助。
Materials
| 0.7 mm diameter burr | Fine Science Tools | 19007-07 | For Screws No. 19010-00 |
| 0.9 mm diameter burr | Fine Science Tools | 19007-09 | |
| ASEPTICO AEU-12C TORQUE PLUS | from Handpiece solution | AEU12C | |
| Bull dog serrifine clump | Fine Science Tools | 18050-28 | |
| CellVizio dual band | Mauna Kea Technologies | ||
| CellVizio single band | Mauna Kea Technologies | ||
| Confocal Microprobe 300 microns (Serie S) | Mauna Kea Technologies | ||
| Custom-made alignment piece | L-shaped (angled at 90 deg) and made of stainless steel with two holes drilled on it, with a 4 mm separation from center to center | ||
| Custom-made mounting bar | The long section piece of the mounting bar should be between 9.4 – 13mm. Fixed to this piece of the mounting bar, position a stainless-steel plate 1.5 cm long and 0.5 cm wide that has two holes drilled separated 4 mm from center to center, the same distance that the L-shaped alignment piece. | ||
| Cyanoacrylate adhesive-Super Glue | |||
| Dumont forceps #5 | Fine Science Tools | 11252-20 | |
| DuraLay Inlay Resin – Standard Package | Reliance Dental Mfg Co. | 602-7395 (from patterson dental) | |
| Fillister Head, Slotted Drive, M1.6×0.35 Metric Coarse, 12mm Length Under Head, Machine Screw | MSC industrial direct co. | 2834117 | |
| Fine Point scissor | Fine Science Tools | 14090-09 | |
| Fluorescein 5% w/w lysine-fixable dextran (2MD) | Invitrogen, USA | D7137 | |
| Halsey smooth needle holder | Fine Science Tools | 12001-13 | |
| Kalt suture needle 3/8 curved | Fine Science Tools | 12050-03 | |
| lab standard stereotaxic, rat and mouse | Stoelting Co. 51704 | 51670 | |
| Methocel 2% | Omnivision GmbH | PZN: 04682367 | Eye ointment to prevent dryness. |
| Mouse Temperature controller, probe (YSI-451), small heating pad-TC-1000 Mouse | CWE Inc. | 08-13000 | |
| PhysioTel F20-EET transmitters | DSI | 270-0124-001 | |
| Robot Stereotaxic, Manipulator Arm, ADD-ON, 3 Axis, LEFT | Stoelting Co.C13 | 51704 | |
| Sel-Tapping bone screws | Fine Science Tools | 19010-10 | |
| Standard Ear Bars and Rubber Tips for Mouse Stereotaxic | Stoelting Co | 51648 | |
| Suture Thread – Braided Silk/Size 4/0 | Fine Science Tools | 18020-40 | |
| Tissue separating microspatula | Fine Science Tools | 10091-121 |
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