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

Bu araştırma Kore Sağlık ve Refah Bakanlığı, Cumhuriyet (: H14C04660000 hibe sayısı) tarafından finanse Kore Sağlık Sanayi Geliştirme Enstitüsü (KHIDI) aracılığıyla Kore Sağlık Teknoloji Ar-Ge Projesi hibe ile desteklenmiştir.

Tüm hayvan deneyleri (1978 revize NIH Yayınları No. 8023) Laboratuvar Hayvanları Bakım ve Kullanım Sağlık Rehberi National Institutes göre yürütülen ve Kurumsal Hayvan Bakım ve Kullanım Komitesi Hayvan Bakım ve Kullanım Komitesi ile yapıldı KIST (Seul, Kore). 1. Hayvan hazırlanması <ol><li> 7 haftalık erkek ICR farelere (veya C57BL / 6) bir grup hazırlayın. </li><li> Fareler homeostazı geri taşıma sonra 3-7 gün boyunca aklimasyonundan sürecinde gidelim. Genel olarak, her bir kafeste...

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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>

Bu protokolde en önemli adım Aβ ve ICV enjeksiyonu olduğunu. Bu protokol, stereotaktik araçlar 11,12 olmayan farelerin ICV bölgesine Aβ enjekte etmek için dizayn edilmiştir. Bir deney başlamadan önce, yerine Aβ bir mavi boya ile uygulama enjeksiyonu ön sürenin yeterli maharet elde etmek için yer almalıdır. Enjeksiyondan hemen sonra, pigment enjeksiyonu düzgün gerçekleştirildi olup olmadığını kontrol etmek gerekir. Dikkatle beyin çıkarmak ve uygun bölge mavi boyalı olup olmadığı...

Alzheimer hastalığı (AD) &#39;in bir patolojik özelliğidir amiloid-P (Aβ) göz önüne alındığında, AD, hayvan modellerinin geliştirilmesi Aβ nöral aşırı ekspresyonu üzerinde odaklanmıştır. Amiloid öncü proteinin (APP) veya presenilin (PS) mutasyonlar Aβ denge bozulmasına ve sonuçta ailesel AD 1 patogenezinde yol, çünkü APP veya PS gen mutasyonlarını içeren fare modelleri genel olarak kabul edilmiştir. Transgenik farelerin geniş arasında prototip fare modelleri şunlardır: TG2576, PDAPP APP / PS1 ve APP23. Beyinde, bu fareler, genellikle Aβ agregasyonu ve sonunda senil plaklar gösterirler; plak oluşumu, öğrenme ve bellek davranış testleri düşük performans gösteren, bu önemli bir bilişsel bozulma takip eder. doğal insan AD patoloji taklit transgenik fareler kullanılarak nesil böylece di ilerlemesini izlemek için bize izin AD araştırma topluma katkıda bulunmuşturolgunun hastalığı. Bu Aβ plaklar geliştirmek için fareler ay sürer ve daha uzun Aβ kaynaklı sinaptik ve ruhsal bozukluk 2,3 göstermek Ancak, transgenik fareler kullanılarak ekonomik değildir ve zaman alıcıdır. Başlangıçta transgenik fare modellerinin eksikliklerin üstesinden gelmek için alternatif olarak geliştirilen, transgenik olmayan modelleri de yaygın olarak farklı avantajlar nedeniyle kullanılır. AD gibi bilişsel bozukluklar, diğer kimyasal ve fiziksel tetiklenebilir ise patojen kaynaklı AD modeller vasıtasıyla-örneğin skopolamin olarak nörotoksik bileşiklerin enjeksiyonundan lezyonların indüksiyonu gibi, beyin içine Aβ direkt enjeksiyonu ile üretilebilir böyle hipokampus, ya da kortikal hasar 4 ile biliş ilgili alanlarda. Ancak, kognitif bozukluk olmayan patojenik indüksiyon doğru AD temel patofizyoloji yansıtmamaktadır; bunun yerine, sadece semptomatik sonuçlarını taklit eder. Buna karşılık, bir patojen kaynaklı AD modeli,46; -injected fare modeli, AD-benzeri davranış anormallikleri gösterebilir kalmayıp Aβ patoloji, ailesel ve sporadik AD tarafından paylaşılan ortak özelliği gösterebilir. Beyin dokusunda Aβ plaklar görselleştirmek için zorluk rağmen, AD soruşturma için cazip kılan Aβ enjekte modelin en büyük avantajı kontrol edilebilirliği olduğunu. Araştırmacılar uyuşturucuyla ilgili çalışmalarda hatalı verilere yol açabilir fare modellerinde bireysel farklılıkları ayıklamak yapabilirsiniz. Zamanında ilaç tedavisi aday ilacın mekanizmasına bağlı olarak etkindir; hazırlamak için, Aβ agregasyon inhibitörü Aβ enjeksiyonundan önce uygulanabilir. Ayrıca, araştırmacılar diğer faktörler sıkıca bireysel farklılıklar da dahil olmak üzere, kontrol edilir, çünkü Aβ enjeksiyonundan sonra ortaya çıkan patojenik dönüşüm Aβ maruz türetilmiştir varsayabiliriz. h Bu protokol, canlı açıklamakumaşla sunulmuştur stereotaktik enstrümanların olmadan Aβ intraserebroventriküler (ICV) enjeksiyon yoluyla normal farelerde bir AD-benzeri fenotipi teşvik. Beyin dokusu ekleme-provoke hasarı en aza indirmek yapısal hasar ve lezyon kaynaklı inflamasyon olasılığını önlemek için şarttır. fare kullanımı tecrübeli eksikliği beklenmedik bir nöronal yaralanmaya yol açar. Ayrıca, enjeksiyon sırasında elde edilecek uygun açı ve derinlik sağlayacak teknikleri sık sık hatalar aşmak için özellikle önemlidir. ICV enjeksiyonundan ayrıntılı, canlı bir açıklama ile birlikte, aşağıdaki protokol ile üretilen modelin güvenilirliği, aşağıdaki bölümlerde açıklanmaktadır. Aşağıdaki protokol böylece sonuçta AD toplum için anlamlı bir keşif yol açabilecek bir Steppingstone sağlayarak, AD araştırmaya katkıda güvenilir ve kolay anlaşılır bir araç olabilir.

<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.

Bu bölüm, Aβ agregasyon ve hafıza noksanlıkları Y-labirenti değerlendirme teyidi elde edilebilir sonuçlarının örneklerini göstermektedir. Tam uzunlukta Aβ kullanılarak 42 amino asit, Aβ monomerler, oligomerler karışımı ve fibrillerinin (Şekil 3) (1-42) peptidi üretilmiştir. HFIP kaynaklı monomerization adım sayesinde nispeten homojen monomerler (Lane B) ürün elde edilmiştir. 7 gün kuluçkalamadan sonra, Aβ agrega (hat c), çeşitli boyutlarda...

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

Amiloid-β (Aβ) hayvan modeli tanımlanmış bir miktar ve Aβ parçalarının türlerinin yönetimini sağlayan ve her çalışma grubu içinde bireysel farklılıkları azaltır -injected. Bu protokol, normal farelerde Alzheimer-benzeri davranış bozukluğu üretimini sağlayan, stereotaktik araçlar olmaksızın Aβ intraserebroventriküler (ICV) enjeksiyonu anlatılmaktadır.

Normal Farelerde Amiloid-β Peptitler intraserebroventriküler enjeksiyon Akut Alzheimer gibi Bilişsel Açıkları tetikleyebilecek

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.

Research

JoVE Journal

Neuroscience

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Kalıcı ve yaygın Nöronal İletim için Yenidoğan Fare Beyin intraserooroventriküler Viral Enjeksiyon

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

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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 ...

Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

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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

Bir sıçan modelinde deneysel subaraknoid kanamayı indüklemek için kiazmatik öncesi, tek otolog kan enjeksiyonu

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

Açık Uçlu İşbirlikçi Öğrenmede Beyinler Arası Senkron: FNIRS-Hyperscanning Çalışması

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

FNIRS Hiper Tarama Çalışmasında Beyinler Arası Senkronizasyon Nasıl Hesaplanır ve Doğrulanır

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

fNIRS-Hipertarama Verilerindeki Yönlü Bilgi Akışının Kısmi Dalgacık Dönüşümü Tutarlılık Yöntemi Kullanılarak Ölçülmesi

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

Yüksek İşlevli İnme Hastalarında Bilişsel Eksiklikleri Tespit Etmek için Çift Görevli Stroop Paradigması

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

Farelerin Motor Korteksinde Hücre Dışı Elektrofizyolojik Kayıt

Multichannel Extracellular Recording in Freely Moving Mice

Yazar Spotlight: Serbestçe Hareket Eden Farelerde Nöronal Aktiviteyi İncelemek için Çok Kanallı Hücre Dışı Kayıttaki Gelişmeler 

Serbestçe hareket eden farelerde çok kanallı hücre dışı kayıt

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