Name: intracerebroventricular injection of amyloid-&#946; peptides in normal mice to acutely induce alzheimer-like cognitive deficits
Uploaded: 2016-03-16T13:00:00
Description: Watch this Scientific Journal Video about Intracerebroventricular Injection of Amyloid-Î² Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits at JoVE.com

这项研究是由通过韩国保健产业振兴院（KHIDI），由卫生部和福利，韩国（：H14C04660000授权号）资助的韩国保健技术研发项目的资助。

所有的动物实验均按照健康指南实验动物的护理和使用的国家机构进行（NIH出版物号8023，修订1978），并与机构的动物护理和使用委员会的动物护理和使用委员会KIST（韩国首尔）。 1.动物的制备<ol><li>准备一组7周龄雄性ICR小鼠（或C57BL / 6）。 </li><li>让老鼠经过驯化过程3-7天运输后恢复动态平衡。在一般情况下，把一组4-5只在每个笼子并予以保持在22-23℃和40％湿度，用12/12小时亮/暗循环。?...

<ol>
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V., 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=Amelioration+of+Alzheimer's+disease+by+neuroprotective+effect+of+sulforaphane+in+animal+model.">Amelioration of Alzheimer's disease by neuroprotective effect of sulforaphane in animal model.</a> Amyloid. 20, 7-12 (2013).</li><li>Jackson, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VTE+on+an+elevated+T-maze.">VTE on an elevated T-maze.</a> J Comp Psychol. 36, 9 (1943).</li><li>Kim, H. Y., 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=Taurine+in+drinking+water+recovers+learning+and+memory+in+the+adult+APP/PS1+mouse+model+of+Alzheimer's+disease.">Taurine in drinking water recovers learning and memory in the adult APP/PS1 mouse model of Alzheimer's disease.</a> Sci Rep. 4, 7467 (2014).</li><li>Paxinos, G., Franklin, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. 2nd edn. , (2001).</li><li>Ledo, J. H., 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=Amyloid-beta+oligomers+link+depressive-like+behavior+and+cognitive+deficits+in+mice.">Amyloid-beta oligomers link depressive-like behavior and cognitive deficits in mice.</a> Mol Psychiatry. 18, 1053-1054 (2013).</li><li>Laursen, S. E., Belknap, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebroventricular+injections+in+mice.+Some+methodological+refinements.">Intracerebroventricular injections in mice. Some methodological refinements.</a> J Pharmacol Methods. 16, 355-357 (1986).</li><li>Bromley-Brits, K., Deng, Y., Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morris+water+maze+test+for+learning+and+memory+deficits+in+Alzheimer's+disease+model+mice.">Morris water maze test for learning and memory deficits in Alzheimer's disease model mice.</a> J Vis Exp. , (2011).</li><li>Figueiredo, C. P., 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=Memantine+rescues+transient+cognitive+impairment+caused+by+high-molecular-weight+abeta+oligomers+but+not+the+persistent+impairment+induced+by+low-molecular-weight+oligomers.">Memantine rescues transient cognitive impairment caused by high-molecular-weight abeta oligomers but not the persistent impairment induced by low-molecular-weight oligomers.</a> J Neurosci. 33, 9626-9634 (2013).</li><li>Haley, T. J., McCormick, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+effects+produced+by+intracerebral+injection+of+drugs+in+the+conscious+mouse.">Pharmacological effects produced by intracerebral injection of drugs in the conscious mouse.</a> Br J Pharmacol Chemother. 12, 12-15 (1957).</li><li>Harkany, T., 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=Neuroprotective+approaches+in+experimental+models+of+beta-amyloid+neurotoxicity:+relevance+to+Alzheimer's+disease.">Neuroprotective approaches in experimental models of beta-amyloid neurotoxicity: relevance to Alzheimer's disease.</a> Prog Neuropsychopharmacol Biol Psychiatry. 23, 963-1008 (1999).</li><li>Wang, D., 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+allosteric+potentiation+of+nicotinic+acetylcholine+receptors+by+galantamine+ameliorates+the+cognitive+dysfunction+in+beta+amyloid25-35+i.c.v.-injected+mice:+involvement+of+dopaminergic+systems.">The allosteric potentiation of nicotinic acetylcholine receptors by galantamine ameliorates the cognitive dysfunction in beta amyloid25-35 i.c.v.-injected mice: involvement of dopaminergic systems.</a> Neuropsychopharmacology. 32, 1261-1271 (2007).</li><li>Yamada, K., Nabeshima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+Alzheimer's+disease+and+evaluation+of+anti-dementia+drugs.">Animal models of Alzheimer's disease and evaluation of anti-dementia drugs.</a> Pharmacol Ther. 88, 93-113 (2000).</li><li>Cho, S. M., 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=Correlations+of+amyloid-beta+concentrations+between+CSF+and+plasma+in+acute+Alzheimer+mouse+model.">Correlations of amyloid-beta concentrations between CSF and plasma in acute Alzheimer mouse model.</a> Sci Rep. 4, 6777 (2014).</li></ol>

在这个协议中最重要的步骤是Aβ的ICV注射。该协议被设计为注射的Aβ到小鼠ICV区而不立体定向仪11,12。在开始实验之前，实践注射用蓝色染料代替Aβ初步期间应该发生达到足够灵巧。注射后立即，有必要检查颜料喷射是否正确执行。小心取出大脑，并检查相应的区域是否被染成蓝色。着色线应达到，但不超过，该ICV区域的深度。 90％的成功率，这是经过50以上的ICV注射一般能达到的最低限?...

鉴于淀粉样蛋白β（Aβ）是阿尔茨海默氏病（AD）的一个病理标志，AD动物模型的发展集中于Aβ的神经过度表达。因为在淀粉样前体蛋白（APP）或早老（PS）的突变导致Aβ的平衡的干扰，并最终以家族性AD 1的发病机制，涉及APP或PS基因突变的小鼠模型已被普遍接受。之间的宽范围的转基因小鼠的，典型的小鼠模型包括如下：的Tg2576，PDAPP，APP / PS1和APP23。在大脑中，这些小鼠通常显示出Aβ凝聚并最终老年斑;噬菌斑形成后跟显著认知障碍，使得它们显示在学习和记忆的行为测试性能不佳。利用转基因小鼠自然地模仿人类的​​AD病理的产生使我们能够监测迪的发展也由此促成了AD研究会sea​​se。但是，使用转基因小鼠是不经济的和费时的，因为它需要几个月的小鼠发展的Aβ斑块和甚至更长的时间才能显示Aβ诱导的突触或行为异常2,3。最初开发作为替代克服转基因小鼠模型的缺点，非转基因模型通常也由于其独特的优点使用。病原体诱导的AD模型可以通过直接注射的Aβ进入大脑产生，而AD样认知缺陷也可以通过其它化学和物理触发装置 - 如注射如东莨菪碱的神经毒性的化合物，病变的感应在认知相关的领域，如海马，或皮质伤害4。然而，所述非致病性的诱导的认知损害不能准确反映AD的根本病理生理学;相反，它只是模仿其症状的结果。相反，病原体诱导AD模型中，A46; -injected小鼠模型，不仅可以显示AD样行为异常也可以表现出Aβ病理，由家族性和散发AD共享的公共功能。 尽管以可视化的Aβ斑块在脑组织中的困难，这使得它对于AD调查吸引力Aβ注射模型的最大好处是它的可控制性。研究人员可以淘汰在小鼠模型中的个体差异，可能导致错误的数据与毒品有关的研究。及时的药物治疗是根据所述候选药物的机制使能;阐述，Aβ聚集抑制剂可以Aβ注射前被应用。此外，研究人员可以假定致病变换Aβ注射后产生从Aβ的曝光衍生，因为其它因素严格控制，包括个体差异。 在这个协议中，H生动的描述流诱导Aβ经脑室（ICV）注射正常小鼠的AD样表型立体定向仪呈现。减少到脑组织插入挑起损伤是至关重要的，以防止结构损坏和损伤诱导的炎症的可能性。缺乏在鼠标操作技能导致意想不到的神经元损伤。此外，技术，使相应的角度和深度的注射过程来实现是规避失误频频尤其重要。除了ICV注射的详细，生动的说明中，通过以下方案所产生的模型的可靠性在下面的章节也示出。下面的协议可能是一个可靠的，易于理解的工具，有助于AD的研究，从而提供一个跳板，可能最终导致一个有意义的发现为AD的社会。

<table border="0" cellpadding="0" cellspacing="0" width="1106">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">ICR mouse</td>
			<td style="width:155px;">Orientbio</td>
			<td style="width:119px;"></td>
			<td style="width:532px;">male, 6~8 weeks, 27~29 g of body weight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6 mouse</td>
			<td>Orientbio</td>
			<td></td>
			<td>male, 6~8 weeks, 21~23 g of body weight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amyloid-beta1-42</td>
			<td>in house synthesis</td>
			<td>n.a.</td>
			<td>stock concentration: 1 mM/DMSO, injected concentration: 100 &#956;M/10% DMSO and 90% PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ICV injection syringe (26s gauge)</td>
			<td>Hamilton</td>
			<td>80308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Evans blue dye (EBD)</td>
			<td>abcamBIochemicals</td>
			<td>ab120869</td>
			<td>1 % EBD in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gradi-GelTM II Gradient PAGE Analysis Kit</td>
			<td>ELPiS Biotech</td>
			<td>EBS-1056</td>
			<td>15% Gel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precision Plus ProteinTM Dual Xtra Standards</td>
			<td>Bio-Rad</td>
			<td>161-0377</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silver-Staining Kit</td>
			<td>GE-Healthcare</td>
			<td>17-1150-01</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

此部分示出了可以由Aβ聚集和记忆障碍的Y-迷宫评估确认来得到的结果的例子。使用全长Aβ制作（1-42）的42个氨基酸，Aβ单体，低聚物的混合物，和原纤维（ 图3）的肽。通过HFIP诱导monomerization步骤，获得相对均匀的单体（泳道B）。 7天孵化后，Aβ聚集体（泳道C）的不同尺寸的发展。三聚体和四聚体Aβ的寡聚形式中的主导品种。空间工作记忆经由在Y迷宫测试?...

Watch this Scientific Journal Video about Intracerebroventricular Injection of Amyloid-Î² Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits at JoVE.com

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

淀粉样蛋白β（Aβ）-injected动物模型能够确定的数量和Aβ片段的物种的管理，并降低每个研究组内的个体差异。本协议描述Aβ的脑室（ICV）注射立体定向仪，使生产正常小鼠老年痴呆样行为异常。

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, ...

The overall goal of this surgical procedure is to conduct the intracerebroventricular injection of Amyloid beta without stereotactic instruments, and to acutely induce Alzheimer-like behavior abnormalities in normal mice. This method can help answering key questions in the field of neurobiology, such as drug discovery for Alzheimer's Disease. The main benefit of this technique is that we can perform the direct injection of A beta into the intracerebroventricle region of the mice without the help of stereotactic instruments, and it allows us to mimic Alzheimer-like behavior abnormalities in mice without waiting for the aging process.The implications of this technique extend towards therapy or diagnosis of Alzheimer's Disease, because it can be used to study the downstream pathology of Amyloid beta, and screened for drug candidates. Generally, individual new to this method will struggle, because finding the precise location of bregma on animal skull may be difficult. To begin this procedure, make one millimolar A-beta with DMSO.Then, dilute it ten fold in PBS. Next, prepare PBS solution containing 10%DMSO as a vehicle for ICV injection. After that, wrap the microsyringe with a 26-gauge stainless steel needle using Parafilm strips in order to adjust the needle length for depth-controlled injection.Densely wrap the syringe multiple times so that the Parafilm will not be compressed during needle insertion, and the needle length stays constant. Then, cut the layers of Parafilm strips to adjust the needle length to 3.8 millimeters between the needle tip and the Parafilm. Next, wash the syringe twice with 70%ethanol.Subsequently, rinse the syringe with distilled water. Dry it completely in a fume hood for at least 30 minutes, and leave the clean syringe under a UV light for another 20 minutes before injection. In this step, place a seven week old anesthetized mouse on a warm mat.Next, apply sterile PBS drops or ointment to both eyes to prevent corneal dryness during the procedure. Afterward, confirm adequate anesthesia by assessing the toe pinch extensor withdrawal reflex. Then, prepare two mirrors with multiple vertical lines drawn on the surfaces to provide references for the syringe's perpendicular injection.Place one mirror right next to the body of the mouse, and the other in front of the head. Keep M1 parallel to the imaginary midline between the two eyes of the mouse and arrange M2 perpendicular to M1, so that the two planes form a 90 degree angle. Next, spray 70%ethanol on the mouse forehead, and rub it with dry cotton swabs.To locate the bregma, place the thumb and index fingers directly above the eyes and slightly press the forehead down to the extent that both eyes protrude. At the same time, drag the skin of the forehead backwards in order to minimize the skull movement under it. Locate the injection site with a tailor's tape at 2.4 millimeters depth under the skull.Fill the syringe with 10 microliters of A-beta solution or vehicle. Place the syringe so that it is perpendicular to the plane of the injection site. Then, align the reflected images of the syringe in both mirrors with the drawn lines on the mirrors.Begin inserting the needle until the Parafilm wrapping touches the skin. Hold the syringe with one hand, and inject five microliters of the A-beta solution or vehicle with the other hand, slowly over five seconds without pausing, and make sure the syringe remains perpendicular throughout. After completing the injection, wait three to five seconds before removing the syringe.Place the thumb and index fingers back on the head, and hold it down to prevent the skull from any unneccessary movements. Then remove the syringe without tilting. Subsequently, place the mouse on a warm pad for recovery.To confirm if A-beta was injected in the target ICV region, check the trace of the needle insertion in the brain tissue to see if it reached one of the ventricles. If the injection was unsuccessful, exclude that subject's data from analysis of the experimental results. In this experiment, the full-length A-beta peptide of 42 amino acids was used to produce a mixture of A beta monomers, oligomers, and fibrils.Through the HFIP induced monomerization step, relative homogenous monomers were obtained. After the seven day incubation, diverse sizes of A-beta aggregates were developed, in which trimers and tetramers were the dominant species among the oligomeric forms of A-beta. In this study, spacial working memory was assessed in the A-beta injected mice in the Y-maze test, in which the sequence of arm choices and the number of total arm entries were recorded while each mouse was allowed to freely explore the maze.The A-beta injected mouse group showed significantly lower alternation rates, indicating the development of cognitive deficits. Once mastered, this technique can be done in 20 minutes, including anesthetic procedures, if it is performed properly. While attempting these procedures, it is important to remember to make the needle perpendicular to the plane of injection.After its development, this technique paved the way for researchers in the field of drug discovery of Alzheimer's Disease to explore in vivo screening of drug candidates in these easily accessible Alzheimer's rodent models. After watching this video, you should have a good understanding of how to conduct intracerebroventricular injection without stereotactic instruments.

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Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

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The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In ...

Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model

视交叉前，单次注射自体血以诱导大鼠模型中实验性蛛网膜下腔出血

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely ...

Inter-Brain Synchrony in Open-Ended Collaborative Learning: An fNIRS-Hyperscanning Study

开放式协作学习中的脑间同步：fNIRS超扫描研究

fNIRS hyperscanning is widely used to detect the neurobiological underpinnings of social interaction. With this technique, researchers qualify the ...

How to Calculate and Validate Inter-brain Synchronization in a fNIRS Hyperscanning Study

如何在fNIRS超扫描研究中计算和验证脑间同步

The dynamics between coupled brains of individuals have been increasingly represented by inter-brain synchronization (IBS) when they coordinate with each ...

Measurement of the Directional Information Flow in fNIRS-Hyperscanning Data using the Partial Wavelet Transform Coherence Method

使用偏小波变换相干法测量 fNIRS-超扫描数据中的定向信息流

Social interaction is of vital importance for human beings. While the hyperscanning approach has been extensively used to study interpersonal neural ...

Dual-Task Stroop Paradigm for Detecting Cognitive Deficits in High-Functioning Stroke Patients

检测高功能卒中患者认知缺陷的双任务斯特鲁普范式

General clinical cognitive assessment scales are not sensitive enough to cognitive impairment in high-functioning stroke patients. The dual-task ...

小鼠运动皮层中的细胞外电生理记录

Multichannel Extracellular Recording in Freely Moving Mice

作者聚焦：用于研究自由移动小鼠神经元活动的多通道细胞外记录的进展 

自由移动小鼠的多通道细胞外记录

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by ...