低聚淀粉样蛋白-β的立体灌注到小鼠海马
Summary
Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.
Abstract
阿尔茨海默氏病是一种神经退行性疾病，影响人口老化。该疾病的一个主要神经病理学特征是过度产生淀粉样蛋白β的和的β-淀粉样斑块中的患病个体的脑区的沉积。多年来科学家已经产生的试图复制的淀粉样蛋白β病理学众多阿尔茨海默氏病小鼠模型。不幸的是，在小鼠模型中只选择性地模仿疾病特征。神经元死亡，在早老性痴呆病患者的脑中一个显着的效果，是明显缺乏在这些小鼠。因此，我们和其他人已采用直接注入淀粉样蛋白β的可溶性低聚物质的方法 – 淀粉样蛋白β的已被证明是最毒的神经元的形式 – 立体定向到大脑。在本报告中，我们利用雄性C57BL / 6J小鼠文件增加一个选择脑区域淀粉样蛋白β水平的这种手术技术。该输液目标是海马的齿状回，因为这大脑结构，沿与由胆碱能电路连接在所述基底前脑，表示的变性疾病的领域之一。提升β-淀粉样蛋白在通过立体输液齿状回的结果显示增加神经元丢失在齿状回1周内，虽然在布罗卡的斜角带垂直肢体同时增加细胞死亡和胆碱能神经元缺失的基底前脑。这些影响观察到2周。我们的数据表明，目前的β-淀粉样蛋白输注模型提供了一种替代的小鼠模型在一个短期的基础上处理区域特定神经元死亡。该模型的优点在于，β淀粉样蛋白可在空间和时间的方式升高。
Introduction
淀粉样蛋白斑沉积，这是由淀粉样蛋白-β（Aβ1-42）的，是阿尔茨海默氏病（AD）的病理学的关键特征。大量研究表明，过高或毒性水平的重组低聚Aβ1-42引起神经元死亡，突触营养不良，损失和功能障碍;以及学习记忆障碍1-4。受影响的脑区域包括海马，皮质，并如基底前脑和扁桃体5,6-皮层下结构。迄今为止，有试图模拟的Aβ1-42病理学的AD的多个转基因小鼠模型。根据应变这些动物被证明是在审查AD的选择病理特征非常有用。不幸的是，除2转基因品系，APP23和5XFAD，这些小鼠从未完全复制的神经元损失，AD的一个关键方面。即使在APP23和5XFAD观察神经元的损失，神经元死亡，安装前后VED是微妙的，年龄依赖性，孤立的几个选择地区7,8。
直接注入低聚的Aβ1-42入野生型小鼠的大脑提供的体内模型它复制amyloidopathy 1,9,10的神经元死亡方面极好。不像通常用于转基因小鼠模型的低聚的Aβ1-42输注模型是理想的急性升降的Aβ1-42的水平在空间和时间的方式。使用野生型小鼠对这种模式的优点避免从在转基因小鼠系引入的突变势补偿或副作用。过去的研究已经表明，注入的Aβ1-42的毒性水平进海马引起神经元死亡在注射部位的附近1周1次内。此外，与观察一致，即Aβ1-42为胆碱能神经元11的基底前脑的有毒胆碱能神经元（BFCN）人群中伸出的海马在7-14天以下的β-淀粉样蛋白注射1,10小鼠下降20-50％，从而有效的隔离神经回路中的脑组织考试。因为BFCN项目同侧海马12的齿状回，在大多数情况下控制/车辆和低聚的Aβ1-42溶液可对大脑允许将左和右半球1之间进行比较的任一侧被注入。
在这份报告中，我们将提供成人野生型C57BL / 6J小鼠进行详细的手术和注射方法。这种小鼠品系被选中，是因为它在研究广泛应用。从技术上讲，任何脑区域可以针对输液，但在这里，我们将使用海马的齿状回作为目标来说明该技术。
Protocol
注：对于所有的动物实验，体制和照顾和使用实验动物的国家准则随访。 1.准备手术器械和解决方案手术高压灭菌全不锈钢手术器械。 通过稀释200标准的无水乙醇，用无菌分子级去离子蒸馏水制备70％的乙醇。 装上29克针的汉密尔顿注射器。通过反复制定和喷射超纯水1分钟清洁Hamilton注射器和针头的内部。重复此过程，用70％的乙醇。 取下汉密尔?…
Representative Results
制备人重组低聚Aβ1-42的本发明的方法产生的可溶性低聚物质选自单体，二聚体，三聚体和四聚体（ 图1A）的。这些低分子量的Aβ1-42物种，而不是原纤维和斑块，已被证明在许多设置是最毒的神经元1,4,9,17-19。以确定是否低聚的Aβ1-42诱导的神经元死亡的鼠脑的Aβ1-42（4微升100μM的原液）被注入海马DG在9个月大的野生型C57BL一个半球/ 6J小鼠。一个…
Discussion
为了实现成功的Aβ1-42注射实验者或外科医生必须：1）使用无菌技术; 2）正确识别的准确坐标兴趣大脑区域; 3）能够正确地固定在鼠标的立体框架与夷为平地，在AP和ML轴的大脑; 4）必须与精密操作的微操作的能力; 5）确保适当的术后护理。如果遵循这些关键步骤，鼠标应该生存下去，没有观察到感染的手术。
符合体外研究中 ，我们和其他人已经表?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作是由神经疾病研究所的支持和中风授予NS081333（至CMT），阿尔茨海默氏症协会授予NIRG-10-171721和心理健康补助MH096702研究所（UH到），以及国家老龄问题研究所资助的阿尔茨海默氏症研究中心在哥伦比亚大学授予试点AG008702（以YYJ和JB）。
Materials
| Ketamine HCl (100mg/ml) | Henry Schein Medical | 1049007 | 100 mg ketamine per 1 kg animal |
| Xylazine (20mg/ml) | Henry Schein Medical | not available | 10 mg xylazine per 1 kg animal |
| Buprenex (0.3mg/ml) | Henry Schein Medical | 1217793 | 0.1 mg buprenex per 1 kg animal |
| Aβ1-42 | David Teplow/UCLA | not available | 100 μM; This amyloid was used in the paper |
| Aβ1-42 | Bachem | H-1368 | Can be used in place of amyloid from the Teplow lab |
| Aβ1-42 | American Peptide | 62-0-80B | Can be used in place of amyloid from the Teplow lab |
| Scrambled Aβ1-42 | American Peptide | 62-0-46B | Can be used as control peptide for comparing Aβ1-42 |
| NU4 Antibody (Oligomeric Amyloid Antibody) | Gift from William Klein/Northwestern U. | not available | 1:2000 dilution |
| Anti-Amyloid Oligomeric Antibody (Polyclonal Rabbit) | EMD Millipore | AB9234 | May be used in place of Nu4; needs to be tested by the end user |
| 6E10 Antibody (Monoclonal Mouse) (Amyloid Antibody) | Biolegend | sig-39320 | 1:1000 dilution |
| ChAT Antibody (Polyclonal Goat) | Millipore | AB144P | 1:100 dilution |
| DeadEnd Fluorometric TUNEL system | Promega | G3250 | Follow manufacturer's directions for use |
| Prolong Gold Antifade Reagent with DAPI | Invitrogen | P36935 | Use when coverslipping slides |
| Fluorogold | Fluorochrome, LLC | not available | 2% solution |
| Absolute Ethanol (200 proof) | Fisher Scientific | BP2818-4 | For making 70% ethanol for sanitizing and disinfecting |
| Novex 10-20% Tricine gel | Life Technologies | EC6625BOX | For separating Aβ1-42 |
| Novex Tricine SDS Running Buffer (10X) | Life Technologies | LC1675 | For running 10-20% Tricine gels |
| Novex Tris-Glycine Transfer Buffer (25X) | Life Technologies | LC3675 | For transferring 10-20% Tricine gels |
| SuperSignal Western Blot Enhancer | Thermo Scientific | 46640 | For enhancing Aβ1-42 signal; follow manufacturer's protocol |
| Protran BA79 Nitrocellulose Blotting Membrane, 0.1 μm | GE Healthcare Life Sciences | 10402088 | For transferring 10-20% Tricine gels |
| Xcell SureLock Mini-Cell | Life Technologies | EI0001 | Electrophoresis aparatus for running 10-20% Tricine gels |
| GenTeal Lubricant Eye Gel | Novartis | not available | For keeping the mouse eyes moist during surgery; can be found in local pharmacy stores |
| Refresh Optive Lubricant Eye Drops | Allergan | not available | For keeping the mouse eyes moist during surgery; can be found in local pharmacy stores; Can be used in place of GenTeal |
| Betadine | Stoelting | 50998 | For sanitizing and disinfecting |
| Round/Tapered Spatula | VWR | 82027-490 | For opening animal mouth |
| Bulldog Serrefines Clamps (Jaw Dims. 9X1.6mm; Length 28mm) | Fine Science Tools | 18050-28 | Optional; For keeping scalp skin apart during injection |
| Straight Fine Scissors (Cutting edge 25mm; Length 11.5cm) | Fine Science Tools | 14060-11 | For cutting scalp |
| #3 Scalpel Handle | Fine Science Tools | 10003-12 | |
| #11 Surgical Blade | Fine Science Tools | 10011-00 | For making scalp incision |
| Student Standard Pattern Forcep (Tip Dims. 2.5×1.5mm; Length 11.5cm) | Fine Science Tools | 91100-12 | For holding scalp closed during suturing |
| Trimmer Combo Kit | Kent Scientific | CL9990-1201 | For shaving hair |
| T/Pump Warm Water Recirculator | Kent Scientific | TP-700 | For warming animal during surgery |
| Resusable Warmining Pad (5" x 10") | Kent Scientific | TPZ-0510FEA | For attaching it to the T/Pump warm water recirculator to warm the animal during surgery |
| Cordless Micro Drill | Stoelting | 58610 | Use 0.8mm steel burrs to drill holes in the skull |
| Lab Standard Stereotaxic Instrument with Mouse & Neonatal Rat Adaptor | Stoelting | 51615 | |
| Just for Mouse Stereotaxic Instrument | Stoelting | 51730 | Can use this in place of Stoelting Cat. #51615 |
| Quintessential Stereotaxic Injector | Stoelting | 53311 | |
| Dry Glass Bead Sterilizer | Stoelting | 50287 | For sterilizing stainless steel instruments |
| Sterile Surgical Drape (18" x 26") | Stoelting | 50981 | |
| Hamilton Syringe 50 ml, Model 705 RN SYR, NDL | Hamilton Company | 7637-01 | For brain injection; use different syringes for different solutions |
| 29 Gauge Needle, Small Hub RN NDL | Hamilton Company | 7803-06 | For attaching to the Hamilton syringe for brain injection |
| 1 ml BD Tuberculin Syringes | VWR | BD309659 | For administering anesthesia and saline |
| 30 Gauge Needle (0.5") | VWR | BD305106 | For administering anesthesia and saline |
| Portable Electronic CS Series Scale (Ohaus) | VWR | 65500-202 | For weighing animals to determine anesthesia dose |
| Hot plate (Top Plate Dims. 7.25×7.25in) | VWR | 47751-148 | For warming animals post-surgery |
| Sofsilk Silk Suture C-1 Cutting 3/8, 12 mm | Covidien | S1173 | For closing wound |
| Vetbond Tissue Adhesive (3M) | Santa Cruz Biotechnology | sc-361931 | Optional: for aiding in wound closure; Use with suture. |
| Cotton-Tipped Wooden-Shaft Sterile Applicators | Fisher scientific | 22-029-488 | For cleaning and drying surgical wound |
| Fisherbrand Superfrost Plus Microscope Slides | Fisher Scientific | 12-550-15 | For collecting brain sections |
| VWR Micro Cover Glass 24 X 50 mm | VWR | 48393241 | For mounting microscope slides |
| Thermo Scientific Nalgene Syringe Filter 0.2 μm | Fisher Scientific | 194-2520 | For sterilizing saline solution |
| Sterile dual tip skin markers by Viscot Medical | Medline | VIS1422SRL91 | For marking coordinates on the skull |
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Citazione di questo articolo
Jean, Y. Y., Baleriola, J., Fà, M., Hengst, U., Troy, C. M. Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus. J. Vis. Exp. (100), e52805, doi:10.3791/52805 (2015).