Name: stereotaxic infusion of oligomeric amyloid-beta into the mouse hippocampus
Uploaded: 2015-06-17T15:00:00.000+00:00
Duration: 8 min 16 s
Description: Watch this Scientific Journal Video about Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus at JoVE.com

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）。

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<ol><li>高压灭菌全不锈钢手术器械。 </li><li>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。 </li><li>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。 <ol><li>取下汉密尔顿注射器的活塞和针头。空气干燥在层流罩O / N的部分。 </li><li>照射的针头和注射器用紫外线灯使用前30分钟。 </li></ol></li><li>通过在分子级水中溶解的NaCl至最终重量的0.9％的体积浓度制备盐水溶液。 Sterili通过0.2微米的微孔过滤器过滤它泽解决方案。 </li></ol> 2.准备低聚Aβ1-42 <ol><li> Monomerize，重悬重组人的Aβ1-42在DMSO中的5mM完全按法"和其他13的出版物中描述。 </li><li>稀释5毫米Aβ1-42用无菌（1X）PBS以100μM在手术前一天。 </li><li>通过研磨混合解决方案很好。 </li><li>孵育的Aβ1-42溶液12小时，在4℃。 注意:控制解决方案，如在PBS稀释DMSO或加扰/反向的Aβ1-42肽应准备方式相同的Aβ1-42。 </li></ol> 3.确定注射坐标<ol><li>使用成年小鼠脑图谱来确定确切的前-后（AP），内侧-外侧（ML），和背腹（DV）的坐标为感兴趣14的大脑区域。 没有TE:为了说明我们选择了海马齿状回为目标与前囟门以下坐标技术:AP，-2.00毫米; ML，±1.3毫米; DV，-2.2毫米。该AP值前面的负号表示它是前囱为2.00mm后。对ML值之前的±符号表示从中心左，右方向。最后，将DV前的负号表示它是从大脑表面腹侧移动。 </li></ol> 4.立体框架安装<ol><li>佩戴外科口罩，无尘实验室外套，和无菌手术手套。 </li><li>擦拭立体定位仪和鼠标适配器，用70％的乙醇。 </li><li>躺在柜台无菌手术铺巾。 </li><li>放置在立体定位仪上的无菌手术单上面鼠标适配器。 </li><li>擦拭鼠标加热垫，用70％的乙醇。定位加热垫在立体框架床。使用通用labora保守党标签磁带到磁带上下加热垫在鼠标适配器床。 <ol><li>根据制造商的指示装上加热垫，以温水再循环器泵。 </li><li>打开温水再循环器泵和设定温度至37℃，然后等待，直到它达到的温度。 </li></ol></li><li>通过拧到根据制造商的方向电动垂直轴注射贴上50微升汉密尔顿注射器用29克针到立体框架。 </li><li>打开珠灭菌，并等待，直到它达到制造商的预设温度。 注:这是用来消毒的动物之间的不锈钢仪表。这需要仪器20秒，珠灭菌接触。 </li><li>打开热板，并将其设定为42℃，顶上放置一个干净的空笼子。 </li><li>而汉密尔顿注射器固定在立体框架上使用的机动立体我njector绘制了Aβ1-42的解决方案。 注意:绘制了超过4微升溶液到注射器，然后用立体定位喷射器设置注入量。根据制造商的说明书进行操作的喷射器。 </li></ol> 5.动物的制备<ol><li>确定鼠标的重量用电子秤。 </li><li>通过注入腹腔（IP）麻醉小鼠氯胺酮/甲苯​​噻嗪鸡尾酒在100毫克/千克氯胺酮和10毫克/公斤甲苯噻嗪。由脚趾捏反射损耗监测麻醉深度。 </li><li>之后鼠标镇静采取理发器，刮胡子它的头，露出的皮肤在颅骨。 </li><li>将鼠标上的立体定位床的顶部的加热垫的顶部。 </li><li>用锅铲开嘴，把门牙牙齿镇守内线。固定突出件在鼠标的表面上。夹紧它轻轻放下，而不是太紧。 </li><li>颇似和灰的双边横杆耳朵进外耳道，以确保头。移动每个横梁，直到它击中头骨，然后转动螺丝将其锁定。 <ol><li>为了确保头部固定用你的食指轻轻向下推头。如果头部被正确地固定它不会发出或按下时移动。 </li></ol></li><li>滴一滴眼霜或眼滴的眼睛，让他们湿润。确保眼睛是整个手术过程中的湿润。 </li><li>消毒手术部位用消毒棉签聚维酮碘溶液适用于皮​​肤在颅骨，其次是70％的乙醇。执行交替优碘和70％乙醇清洗2次以上。 </li></ol> 6.手术和输液<ol><li>用手术刀做一个2-3毫米的切口，在头皮中线，然后用直细剪刀切口线延伸至1.0-1.5厘米，以暴露矢状缝颅骨，地标，前囟，和lambda■哪些立体定位坐标基础上的。使用微夹具，保持皮肤分开。 注:前囟门和lambda位置在小鼠大脑图谱"鼠脑立体定位坐标"14解释。 </li><li>取棉签，浸泡在无菌盐水，并用它来清洗颅骨。然后，用干净的干棉签干燥头骨。 </li><li>使用无菌细笔以纪念前囟门点。使用立体显微使得针的尖端刚好触及前囟点来定位Hamilton注射器。 <ol><li>记录的DV坐标。 </li><li>要检查头骨的AP轴是水平移动汉密尔顿注射器向后，使针尖触及的lambda点。 </li><li>记录的DV位置。确保DV坐标前囟和lambda是在0.5毫米。 注意:我们的目标是最小化前囟和lambda之间的DV差。 </li></ol></li><li>使用Micromanipulator到Hamilton注射器针尖返回到前囟点。 </li><li>记录起始位置AP。 </li><li>减去2.00毫米从起始坐标AP计算AP结束位置。 </li><li>使用显微重新定位汉密尔顿注射器针头到最后的AP坐标。 </li><li>记录当前坐标ML。 </li><li>计算左，右结束ML坐标加上或减去1.3毫米从起始ML坐标。 </li><li>使用显微汉密尔顿注射器针头移到要么结束ML坐标。 <ol><li>触针尖在两个结束ML坐标颅骨的表面上，并记录的DV坐标，并确保该值是在0.5毫米。 注意:我们的目标是最小化两个结束ML坐标之间的的DV差。 </li><li>而在一个结局协调拉动针上0.5-1厘米以上的头骨。以无菌细笔，以纪念在日点头骨的E面。重复的头骨对侧此步骤。 </li></ol></li><li>摆动汉密尔顿注射器明确的出路。 </li><li>附加0.8毫米钻头的钻。取的钻头用双手，该表用于稳定性的表面上设置弯管，并略定位钻头上方的左侧或右侧的ML坐标Sharpie笔点。激活钻，下钻尖到颅骨表面引入头骨一个洞。重复对侧此步骤。 注意:有些出血可能由新引进的孔出现。如果有出血然后用干净的干棉签轻拍血液。 </li><li>移动汉密尔顿注射器放回原位了无论是新推出的ML孔。降低汉密尔顿针刚刚过去的头骨。在这一点上要小心，不要穿刺大脑。为了确保针头已通过颅骨把你的食指，轻轻推针对头骨进行河畔Ë针不会退出孔。 </li><li>记录起始DV坐标。 </li><li>计算减去从起始坐标DV2.2毫米结束DV坐标。 </li><li>降低针下来到结束DV坐标。 </li><li>激活立体注射器泵将4微升的Aβ1-42的成齿状回以0.5微升/分的速率。 </li><li>当输液完成让针头留在原地另外的1分钟，以最小化的溶液回流出注射部位。 </li><li>移动针到大脑的另一侧，并重复步骤6.13-6.18。 </li></ol> 7.动物搬运和术后护理<ol><li>拧耳朵酒吧和面部/头套。从该装置中取出的小鼠。 </li><li>用学生的标准模式钳拉近头皮，然后用密封缝合伤口。 </li><li>注射1毫升无菌生理盐水为通过IP鼠标水化。 </li><li>在拍摄对象丁丙诺啡（0.1毫克/公斤）皮下注射，以减轻疼痛。 </li><li>保持鼠标在干净的空笼子上42℃的热板的顶部设置，直到它被唤醒，然后用充足的食物和水返回到其住房笼。 </li><li>通过皮下注射给药丁丙诺啡每12小时3天缓解疼痛。 </li></ol> 8.拆线<ol><li>麻醉小鼠氯胺酮/甲苯​​噻嗪鸡尾酒在100毫克/千克氯胺酮和10毫克/公斤xlyazine通过IP。注:这是手术后7-10天进行。 </li><li>用学生的标准模式镊子和直细剪刀去除缝线。 </li><li>注射1毫升无菌生理盐水为通过IP鼠标水化。 </li><li>保持小鼠在一个干净的空笼上42℃的热板的顶部设置，直到其被唤醒，然后将其与充足的食物和水返回到其壳体笼。 </li></ol> 9.动物牺牲和组织处理<ol><li>麻醉小鼠和p用4％多聚甲醛15 erfuse它，取出大脑，并处理它cryosectioning 16。 注:动物献祭的时间取决于实验者。然而，对于我们的研究中，我们选择了7和14天手术后。 </li></ol>

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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-derived+BDNF+supports+myelin+protein+synthesis+after+cuprizone-induced+demyelination.">Astrocyte-derived BDNF supports myelin protein synthesis after cuprizone-induced demyelination.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 34 (24), 8186-8196 (2014).</li><li>Thakker-Varia, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuropeptide+VGF+is+reduced+in+human+bipolar+postmortem+brain+and+contributes+to+some+of+the+behavioral+and+molecular+effects+of+lithium.">The neuropeptide VGF is reduced in human bipolar postmortem brain and contributes to some of the behavioral and molecular effects of lithium.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 30 (28), 9368-9380 (2010).</li><li>Greferath, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enlarged+cholinergic+forebrain+neurons+and+improved+spatial+learning+in+p75+knockout+mice.">Enlarged cholinergic forebrain neurons and improved spatial learning in p75 knockout mice.</a> The European journal of neuroscience. 12 (3), 885-893 (2000).</li><li>Brashear, H. R., Zaborszky, L., Heimer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+GABAergic+and+cholinergic+neurons+in+the+rat+diagonal+band.">Distribution of GABAergic and cholinergic neurons in the rat diagonal band.</a> Neuroscience. 17 (2), 439-451 (1986).</li><li>Clarke, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+innervation+of+the+rat+dentate+gyrus:+an+immunocytochemical+and+electron+microscopical+study.">Cholinergic innervation of the rat dentate gyrus: an immunocytochemical and electron microscopical study.</a> Brain research. 360 (1-2), 349-354 (1985).</li><li>Garcia-Osta, A., Alberini, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+beta+mediates+memory+formation.">Amyloid beta mediates memory formation.</a> Learning &amp; memory. 16 (4), 267-272 (2009).</li></ol>

作者宣称，他们有没有竞争的财务权益。

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明<...

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。

<table><tbody><tr><td>Ketamine HCl (100 mg/ml)</td><td>Henry Schein Medical</td><td>1049007</td><td>100 mg ketamine per 1 kg animal</td></tr><tr><td>Xylazine (20 mg/ml)</td><td>Henry Schein Medical</td><td>not available</td><td>10 mg xylazine per 1 kg animal</td></tr><tr><td>Buprenex (0.3 mg/ml)</td><td>Henry Schein Medical</td><td>1217793</td><td>0.1 mg buprenex per 1 kg animal</td></tr><tr><td>A&amp;beta;&lt;sub&gt;1-42&lt;/sub&gt;</td><td>David Teplow/UCLA</td><td>not available</td><td>100 &amp;mu;M; This amyloid was used in the paper</td></tr><tr><td>A&amp;beta;&lt;sub&gt;1-42&lt;/sub&gt;</td><td>Bachem</td><td>H-1368</td><td>Can be used in place of amyloid from the Teplow lab</td></tr><tr><td>A&amp;beta;&lt;sub&gt;1-42&lt;/sub&gt;</td><td>American Peptide</td><td>62-0-80B</td><td>Can be used in place of amyloid from the Teplow lab</td></tr><tr><td>Scrambled A&amp;beta;&lt;sub&gt;1-42&lt;/sub&gt;</td><td>American Peptide</td><td>62-0-46B</td><td>Can be used as control peptide for comparing A&amp;beta;1-42</td></tr><tr><td>NU4 Antibody (Oligomeric Amyloid Antibody)</td><td>Gift from William Klein/Northwestern U.</td><td>not available</td><td>1:2,000 dilution</td></tr><tr><td>Anti-Amyloid Oligomeric Antibody&amp;nbsp; (Polyclonal Rabbit)</td><td>EMD Millipore</td><td>AB9234</td><td>May be used in place of Nu4; needs to&amp;nbsp; be tested by the end user</td></tr><tr><td>6E10 Antibody (Monoclonal Mouse) (Amyloid Antibody)</td><td>Biolegend</td><td>sig-39320</td><td>1:1,000 dilution</td></tr><tr><td>ChAT Antibody (Polyclonal Goat)</td><td>Millipore</td><td>AB144P</td><td>1:100 dilution</td></tr><tr><td>DeadEnd Fluorometric TUNEL system</td><td>Promega</td><td>G3250</td><td>Follow manufacturer&#039;s directions for use</td></tr><tr><td>Prolong Gold Antifade Reagent with DAPI</td><td>Invitrogen</td><td>P36935</td><td>Use when coverslipping slides</td></tr><tr><td>Fluorogold</td><td>Fluorochrome, LLC</td><td>not available</td><td>2% solution</td></tr><tr><td>Absolute Ethanol (200 proof)</td><td>Fisher Scientific</td><td>BP2818-4</td><td>For making 70% ethanol for sanitizing and disinfecting</td></tr><tr><td>Novex 10-20% Tricine gel</td><td>Life Technologies</td><td>EC6625BOX</td><td>For separating A&amp;beta;1-42</td></tr><tr><td>Novex Tricine SDS Running Buffer (10X)</td><td>Life Technologies</td><td>LC1675</td><td>For running 10-20% Tricine gels</td></tr><tr><td>Novex Tris-Glycine Transfer Buffer (25X)</td><td>Life Technologies</td><td>LC3675</td><td>For transferring 10-20% Tricine gels</td></tr><tr><td>SuperSignal Western Blot Enhancer</td><td>Thermo Scientific</td><td>46640</td><td>For enhancing A&amp;beta;1-42 signal; follow manufacturer&#039;s protocol</td></tr><tr><td>Protran BA79 Nitrocellulose Blotting Membrane, 0.1 &amp;mu;m</td><td>GE Healthcare Life Sciences</td><td>10402088</td><td>For transferring 10-20% Tricine gels</td></tr><tr><td>Xcell SureLock Mini-Cell</td><td>Life Technologies</td><td>EI0001</td><td>Electrophoresis aparatus for running 10-20% Tricine gels</td></tr><tr><td>GenTeal Lubricant Eye Gel</td><td>Novartis</td><td>not available</td><td>For keeping the mouse eyes moist during surgery; can be found in local pharmacy stores&lt;br /&gt;&amp;#160;&lt;br/&gt;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Refresh Optive Lubricant Eye Drops</td><td>Allergan</td><td>not available</td><td>For keeping the mouse eyes moist during surgery; can be found in local pharmacy stores; Can be used in place of GenTeal</td></tr><tr><td>Betadine</td><td>Stoelting</td><td>50998</td><td>For sanitizing and disinfecting</td></tr><tr><td>Round/Tapered Spatula&amp;nbsp;</td><td>VWR</td><td>82027-490</td><td>For opening animal mouth</td></tr><tr><td>Bulldog Serrefines Clamps (Jaw Dims. 9X1.6 mm; Length 28 mm)</td><td>Fine Science Tools</td><td>18050-28</td><td>Optional; For keeping scalp skin apart during injection</td></tr><tr><td>Straight Fine Scissors (Cutting edge 25 mm; Length 11.5 cm)</td><td>Fine Science Tools</td><td>14060-11</td><td>For cutting scalp</td></tr><tr><td>#3 Scalpel Handle</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>#11 Surgical Blade</td><td>Fine Science Tools</td><td>10011-00</td><td>For making scalp incision</td></tr><tr><td>Student Standard Pattern Forcep (Tip Dims. 2.5x1.5 mm; Length 11.5 cm)</td><td>Fine Science Tools</td><td>91100-12</td><td>For holding scalp closed during suturing</td></tr><tr><td>Trimmer Combo Kit</td><td>Kent Scientific</td><td>CL9990-1201</td><td>For shaving hair</td></tr><tr><td>T/Pump Warm Water Recirculator&amp;nbsp;</td><td>Kent Scientific&amp;nbsp;</td><td>TP-700</td><td>For warming animal during surgery</td></tr><tr><td>Resusable Warmining Pad (5&quot; x 10&quot;)</td><td>Kent Scientific&amp;nbsp;</td><td>TPZ-0510FEA</td><td>For attaching it to the T/Pump warm water recirculator to warm the animal during surgery</td></tr><tr><td>Cordless Micro Drill</td><td>Stoelting</td><td>58610</td><td>Use 0.8 mm steel burrs to drill holes in the skull</td></tr><tr><td>Lab Standard Stereotaxic Instrument with Mouse &amp;amp; Neonatal Rat Adaptor</td><td>Stoelting</td><td>51615</td><td></td></tr><tr><td>Just for Mouse Stereotaxic Instrument</td><td>Stoelting</td><td>51730</td><td>Can use this in place of Stoelting Cat. #51615</td></tr><tr><td>Quintessential Stereotaxic Injector</td><td>Stoelting</td><td>53311</td><td></td></tr><tr><td>Dry Glass Bead Sterilizer</td><td>Stoelting</td><td>50287</td><td>For sterilizing stainless steel instruments</td></tr><tr><td>Sterile Surgical Drape (18&quot; x 26&quot;)</td><td>Stoelting</td><td>50981</td><td></td></tr><tr><td>Hamilton Syringe 50 ml, Model 705 RN SYR, NDL</td><td>Hamilton Company</td><td>7637-01</td><td>For brain injection; use different syringes for different solutions</td></tr><tr><td>29 G Needle, Small Hub RN NDL</td><td>Hamilton Company</td><td>7803-06</td><td>For attaching to the Hamilton syringe for brain injection</td></tr><tr><td>1 ml BD Tuberculin Syringes</td><td>VWR</td><td>BD309659</td><td>For administering anesthesia and saline</td></tr><tr><td>30 G Needle (0.5&quot;)</td><td>VWR</td><td>BD305106</td><td>For administering anesthesia and saline</td></tr><tr><td>Portable Electronic CS Series Scale (Ohaus)</td><td>VWR</td><td>65500-202</td><td>For weighing animals to determine anesthesia dose</td></tr><tr><td>Hot plate (Top Plate Dims. 7.25x7.25 in)</td><td>VWR</td><td>47751-148</td><td>For warming animals post-surgery</td></tr><tr><td>Sofsilk Silk Suture C-1 Cutting 3/8, 12 mm</td><td>Covidien</td><td>S1173</td><td>For closing wound</td></tr><tr><td>Vetbond Tissue Adhesive (3M)</td><td>Santa Cruz Biotechnology</td><td>sc-361931</td><td>Optional: for aiding in wound closure; Use with suture.</td></tr><tr><td>Cotton-Tipped Wooden-Shaft Sterile Applicators</td><td>Fisher scientific</td><td>22-029-488</td><td>For cleaning and drying surgical wound</td></tr><tr><td>Fisherbrand Superfrost Plus Microscope Slides</td><td>Fisher Scientific&amp;nbsp;</td><td>12-550-15</td><td>For collecting brain sections</td></tr><tr><td>VWR Micro Cover Glass 24 X 50 mm</td><td>VWR</td><td>48393241</td><td>For mounting microscope slides</td></tr><tr><td>Thermo Scientific Nalgene Syringe Filter 0.2 &amp;mu;m</td><td>Fisher Scientific</td><td>194-2520</td><td>For sterilizing saline solution</td></tr><tr><td>Sterile dual tip skin markers by Viscot Medical</td><td>Medline</td><td>VIS1422SRL91</td><td>For marking coordinates on the skull</td></tr></tbody></table>

stereotaxic infusion of oligomeric amyloid-beta into the mouse hippocampus

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。

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...

Watch this Scientific Journal Video about Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus at JoVE.com

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

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.

低聚淀粉样蛋白-β的立体灌注到小鼠海马

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

stereotaxic-infusion-oligomeric-amyloid-beta-into-mouse

Research

JoVE Journal

Neuroscience

18.1K 次观看.  Columbia University Medical Center. 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.

视频: Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

立体定向注射的microRNA表达慢病毒的小鼠海马CA1区和行为结果的评估

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

科研

行为学

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

低聚β-淀粉样蛋白的制备 1-42和突触可塑性损伤诱导海马脑片

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

神经科学

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

超声引导下显微注射到小鼠前脑在子宫内在E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

Establishment of a Valuable Mimic of Alzheimer's Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure change. With the increase of AD patients worldwide, the pathology and treatment of the disease has become a focus in the International Pharmaceutical Industry. Thus, the establishment of the animal model to mimic AD in the laboratory is of great importance.
Here, we describe a detailed protocol for establishing a mimic of AD in a rat animal model though intracerebroventricular injection of amyloid beta protein 25-35 (A&#946;25-35) combined with aluminum trichloride (AlCl3) and anterodorsal thalamic nucleus injection of recombinant human transforming growth factor-&#946;1 (RHTGF-&#946;1) to rats. The related markers of AD were measured, including: spatial memory, neuronal structure and substructure, neuronal A&#946;, and neurofibrillary tangles (NFT) production. This rat model demonstrates spatial memory impairment, neuronal structure and substructure pathological changes, neuron intracellular A&#946; burden, and NFT aggregation, and provides a close mimic of the neuronal structure and function disorder to that of clinical AD patients. Thus, the presented AD rat modelprovides a valuable in vivo tool for exploring neuronal function, neuronal pathology, and drug screening of AD.

This is a protocol to mimic Alzheimer&#39;s Disease in rats by evaluation of spatial memory impairment, neuronal pathological changes, neuronal amyloid beta protein (A&#946;) burden, and neurofibrillary tangles aggregation, induced by the injection of A&#946;25-35 combined with aluminum trichloride and recombinant human transforming growth factor-&#946;1.

脑室注射复合淀粉样蛋白β模型建立大鼠阿尔茨海默病的价值模拟

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

特定脑区的兴奋毒性损伤的成年大鼠立体定向手术治疗

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

正常小鼠β淀粉样肽侧脑室注射敏锐地诱导老年痴呆样认知缺陷

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (A&#946;o) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble A&#946;o correlate with memory deficits in AD models and humans. The A&#946;o-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated A&#946;o infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning A&#946;o-induced memory deficits at the onset of AD. Since soluble A&#946;o aggregate relatively fast into insoluble A&#946; fibrils that correlate poorly with the clinical state of patients, soluble A&#946;o are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of A&#946;o and continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of A&#946;o in pre-dementia patients.

To study the effects of A&#946;o in vivo, we developed a model based on repeated hippocampal infusions of soluble A&#946;o coupled with continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of A&#946;o.

神经变性慢性淀粉样蛋白-β低聚物输注的动物模型是由抗体治疗灌注抵消与渗透泵

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It ...

医学

Controlled Infusion of A&beta; Peptide-Specific Antibodies and A&beta; Oligomers into the Rat Hippocampus

Source: Sajadi, A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54215">Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for the continuous infusion of amyloid-beta (A&#946;) peptide-specific antibodies into the hippocampus of a rat. The study employs implanting a bilateral cannula system for a controlled infusion of A&#946; peptide-specific antibodies coupled with the infusion of soluble A&#946; oligomers (A&#946;o) into the rat hippocampus to study the interactions between the oligomers and the antibodies.

将 Aβ 肽特异性抗体和 Aβ 寡聚体受控输注到大鼠海马体中

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

Direct Stereotaxic Brain Infusion of Amyloid-Beta Proteins for an Alzheimer's Disease Model

Source: Jean, Y. Y. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52805">Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus.</a> J. Vis. Exp. (2015)
This video demonstrates the precise stereotaxic infusion of amyloid beta into the dentate gyrus to establish a mouse model of Alzheimer&#39;s disease. Accurate coordinate determination and controlled delivery of amyloid beta proteins result in neuronal degradation, disrupting memory processing and contributing to Alzheimer&#39;s disease development.

低聚淀粉样蛋白-β的立体灌注到小鼠海马

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