封闭系统中脑切片长期培养中精确定位和重复间歇成像的改进辊筒法
Summary
本文介绍了一种改进的滚子管法培养和间歇高分辨率成像啮齿动物脑切片多周, 精确重新定位光刻片。神经元的生存能力和切片形态学保持良好。提供了这种完全封闭的系统使用病毒的细胞类型的特定表达的应用。
Abstract
培养的啮齿动物脑切片有助于研究神经元和胶质细胞在维持许多正常的体内相互作用的环境中的分子行为。从各种转基因小鼠线获得的切片或使用病毒载体表达荧光标记蛋白质或记者在野生型脑切片允许高分辨率成像荧光显微镜。虽然有几种方法已经开发成像脑切片, 结合切片培养的能力, 以执行重复高分辨率成像的特定细胞在活片长期的时间已经提出了问题。当病毒载体被用于外源蛋白的表达时尤其如此, 因为这是最好的在一个封闭的系统, 以保护用户和防止交叉污染。简单的修改对辊筒管脑切片培养方法, 允许重复高分辨率成像的切片在一个封闭的系统多周。在光刻片上培养切片允许使用基准标记快速准确地将舞台重新定位到不同处理前后的同一域中。例示为使用此方法结合特定的神经元染色和表达观察海马切片结构的变化, 荧光蛋白的病毒介导的神经元表达, 以及 cofilin 病理的发展,这是先前观察到的阿尔茨海默氏病 (AD) 的海马反应的切片治疗与低聚体淀粉样β (a) 肽。
Introduction
从啮齿动物脑区分离神经元的原代培养是研究人员用来观察病理性牵连刺激反应的重要工具。然而, 这种研究的缺点是只在2D 和没有他们的神经胶质支持系统的情况下观察神经元。此外, 除非在非常高密度的条件下生长 (640 神经元/mm2或大约16% 的表面积), 在这种情况下, 它不可能跟随树枝状或轴突的随机突起, 而从其细胞体, 海马4周以上的神经元存活能力明显下降1, 限制了游离培养对年龄相关病症的扩展研究。从啮齿动物大脑中制备的切片是一种有吸引力的选择, 它通过维持一个有组织的细胞结构和存活数周或数月来克服这些限制。在切片培养中维持啮齿动物大脑许多不同区域的条件已被描述为2。
两种主要的方法被广泛应用于脑切片的长期培养: 在气界面的膜上培养3或在密封管上培养片, 允许在滚筒孵化器中旋转以提供曝气的4。在膜上培养的切片可以直接成像高分辨率荧光显微镜使用直立显微镜和水浸泡目标5。另外, 在膜上培养的切片也被转移到玻璃底盘, 用倒置显微镜6实现树突棘的良好分辨率。然而, 两种在膜上生长的成像切片方法都是开放系统, 需要进行中等程度的改变, 并且经常使用抗真菌和/或抗生素来防止或减少污染5,6。膜上的切片在 air-medium 界面保持优良的形态学和生存, 但返回到精确的位置在重复成像在高放大率是非常困难的, 除非实验只遵循小小组的细胞表示荧光标记。虽然在膜上生长的切片已使用与病毒介导的表达转基因5,6, 生物安全协议可能需要使用封闭的文化系统, 用于某些病毒载体表达荧光标记的蛋白质和细胞生理学记者。此外, 沉浸目标要求在培养5中遵循的样本之间进行净化。膜界面培养的一个主要应用是结合高分辨率成像与电生理学在单时间点7。
在塑料管内片的滚子管法不允许任何电生理学或高分辨率成像, 不排除片。因此, 这种方法最常被应用到长期研究中, post-fixation 观测已经被做为8。这里描述的是一种利用辊筒培养技术的方法, 但对片上的 drilled-out 管, 只要保持文化, 就可以反复成像。封闭的系统不需要介质变化的成像和利用光刻片提供的基准标记, 允许成像在高放大率, 几天或几周后, 精确的领域以前成像。
我们应用这种方法来检查啮齿动物海马的变化, 这是一个主要的大脑区域, 涉及记忆和学习。啮齿类动物的海马体通常被研究为在认知障碍发展过程中观察到的病理或年龄相关变化的模型9, 如 AD 中发生的那些。我们的方法特别适合于研究随着环境变化而在单个切片内发展的病理变化, 如 a 肽的增加, 这是 AD8的特征。一个病理与人类和啮齿动物 AD 脑是存在的 cofilin 肌动蛋白聚合体和棒, 后者含有束的花丝, 其中 cofilin 和肌动蛋白是在1:1 摩尔比10,11,12. 在 a 治疗后的大鼠海马固定切片上观察到了棒状体, 以及在表达 cofilin-GFP 受缺氧8的活鼠脑切片内, 它们可能会导致 AD 中出现的突触功能障碍。和中风。在这里, 我们用这种新的培养方法观察不同病毒引入的外源嵌合荧光蛋白切片中的时间过程和分布。然后利用 cofilin 记者构造的神经元特异性表达, 跟踪 cofilin 棒和海马切片的聚集病理, 以应对可溶性 a 寡聚体 (ao) 的治疗。
Protocol
动物使用遵循批准的育种和动物使用协议, 符合动物保育和使用的准则, 科罗拉多州立大学。 注: 下面的协议描述了长期孵化和间歇性海马切片成像的制备和培养方法。一个单一的海马切片是连接到一个专门准备光刻片使用等离子凝块, 然后片被密封在平坦的一侧的 drilled-out 辊管, 这是保持在滚筒孵化器。血浆凝块在病毒感染前以溶溶解, 用于荧光蛋白表达和高分辨率成像。一?…
Representative Results
为了确定如何准确地使用基准标记来重新同一字段中的同一单元格, 我们检查了在光刻片 (图 6A) 上生长的切片。神经元被染色与一个重要染料 (100 nM 为2小时; 不染色神经细胞细胞), 它从神经元随着时间的推移而消失, 而不损害细胞25。我们在一个格子正方形中确定了一个基准标记 (图 6a, B</stro…
Discussion
这里描述的滚子管法允许长期培养和高分辨率活体影像切片脑组织。切片技术所应用的一个主要问题是切片的安装和维护。片涂层, 支持切片粘附, 促进切片细化, 通过提高突起的生长和细胞迁移的切片;因此, 我们避免使用这些基板。通过 3-aminopropyltriethoxysilane 的治疗, 将氨基基团插入玻璃上, 提高了切片的粘附性, 但在片上太少或太多的鸡血浆也会导致粘附问题导致切片损失。适当的粘附所需的血?…
Materials
| Bottoms from 15 cm culture dishes | VWR Scientific | 25384-326 | |
| Phillips Head Machine Screws (#10-32) | Ace Hardware | 2.5" long and 3/16" in diameter | |
| Flat Washers #10 | ACE Hardware | ||
| Machine Screw Nuts (#10-32) | ACE Hardware | ||
| Rubber Grommets | ACE Hardware | 5/16", thick; 5/8", hole diameter; 1.125", OD | |
| Polyethylene tubing (5/16"; OD, 3/16"; ID) | ACE Hardware | Cut to 1.8" length | |
| Lock Washer #10 | ACE Hardware | ||
| Drill Press, 5 speed | Ace Hardware | ProTech Model 1201 | |
| Nunclon Delta Flat-Sided Tubes | VWR | 62407-076 | |
| Drill bits, 3 mm, 6 mm and 15 mm | Ace Hardware | Diablo freud brand | Drill bits for cutting plastic. |
| Drill bits for wood, 1.5 cm and 1 mm | Ace Hardware | ||
| Wood file, 1/4" round | Ace Harware | ||
| Spring clips, 16 mm snap holder | Ace Hardware | ||
| Swivel Head Deburring Tool, 5" | Ace Hardware | 26307 | |
| Adhesive Silicone Sheet (Secure Seal) | Grace Bio-Labs | 666581 | 0.5 mm Thickness |
| 6 mm hole punch | Office Max | ||
| 12 mm hole punch | thepunchbunch.com | ||
| 70% Ethanol | |||
| Phototeched Coverslips, 12 mm diameter | Bellco Glass, Inc. | 1916-91012 | |
| Bunsen Burner | |||
| Absolute Ethanol | |||
| Nanopure Water | |||
| 3-aminopropyltriethoxylane | Sigma-Aldrich | A3648 | |
| Acetone | Sigma-Aldrich | 179124 | |
| #5 Dumont Forceps | Fine Science Tools | 11251-30 | |
| McIlwain Tissue Chopper | Ted Pella, Inc. | 10180 | |
| Double Edge Razor Blades | Ted Pella, Inc. | 121-6 | |
| Whatman Filter Paper | VWR | 28450-182 | Cut into 5.8 cm diameter circles |
| Poly-chloro-trifluoro-ethylene (Aclar) | Ted Pella, Inc. | 10501-10 | Cut into 5.8 cm diameter circles |
| #21 Surgical Blade | VWR Scientific | 25860-144 | |
| #5 Dumont Forceps | Fine Science Tools | 11251-30 | |
| Spatula, stainless with tapered end | VWR | 82027-518 | |
| Gey's Balanced Salt Solution | Sigma-Aldrich | G9779 | |
| Glucose | ThermoFisher Scientific | 15023-021 | 25% (w/v) Solution, 0.2 mm filter sterilized |
| Chicken Plasma | Cocalico Biologicals | 30-0300-5L | Rehydrate in sterile water, centrifuge at 2500 x g 30 min at 4 °C, quick freeze aliquots in liquid nitrogen and store at -80 °C. |
| Thrombin, Bovine | Fisher | 60-516-01KU | 150 units /ml in GBSS/Glucose, quick freeze aliquots in liquid nitrogen and store at -80 °C. |
| Cell Roller System | Bellco Biotech | SciERA | |
| Roller Incubator | Forma | Model 3956 | |
| N21-MAX | ThermoFisher Scientific | AR008 | |
| Pen/Strep (100X) | ThermoFisher Scientific | 15140122 | |
| 200 mM Glutamine | ThermoFisher Scientific | 25030081 | |
| Glucose | ThermoFisher Scientific | 15023-021 | 25% (w/v) Solution, 0.2 mm filter sterilized |
| Neurobasal A | ThermoFisher Scientific | 10888-022 | Complete Medium: 48 mL Neurobasal A, 1 mL N21-MAX, 0.625 mL 200 mM Glutamine, 0.180 mL 25% Glucose, 0.250 mL 100x pen/strep. |
| Third generation lentivirus packaging | Life Technologies | K4975-00 | |
| 159 K cutoff centrifugal filters (Centricon) | EMD Millipore | ||
| Lentiviral cloning system (InFusion) | Clonetech | ||
| Plasmids 30323, 50856, 51279 | Addgene | ||
| Neuronal cell viability dye (NeuO) | Stemcell technologies | 1801 | Thaw once and quick freeze in 4 µL aliquots. Store at -20 °C |
| Inverted microscope | Olympus | IX83 | |
| Microscope objectives | Olympus | air: 4X, 20; oil: 40X, 60X, | |
| Spinning disc confocal system | Yokagawa | CSU22 | |
| Microscope EMCCD camera | Photometrics | Cascade II | |
| Linear encoded (x,y), piezo z flat top stage | ASI | ||
| Microscope lasers and integration | Intelligent Imaging Innovations | ||
| HEK293T cells | American Type Culture Collection | CRL-3216 | |
| Human Plasmin | Sigma Aldrich | P1867 | 0.002 U/mL in 0.1% bovine serum albumin (0.2 mm filter sterilized), quick freeze in liquid nitrogen and store at -80 °C. |
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Citer Cet Article
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