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

이 연구는 한국의 보건 복지부, 공화국 (: H14C04660000 허가 번호)에 의해 투자 한국 보건 산업 진흥원을 통해 한국 의료 기술 R &amp; D 프로젝트의 기금에 의해 지원되었다.

모든 동물 실험 (1978 개정, NIH 간행물 번호 8023) 실험 동물의 관리 및 사용을위한 건강 가이드의 국립 연구소에 따라 수행과의 기관 동물 관리 및 사용위원회의 동물 관리 및 사용위원회와 함께했다 KIST (서울, 한국). 1. 동물 준비 <ol><li> 7 주령의 수컷 ICR 마우스 (또는 C57BL / 6) 군을 준비한다. </li><li> 쥐가 항상성을 복원 전송 ~ 7 일 동안 적응 과정을 통해 가자. 일반적으로, 각 장에 4-5 마우스 ...

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이 프로토콜에서 가장 중요한 단계는 Aβ의 ICV 주입입니다. 이 프로토콜은 정위기구 (11, 12)없이 마우스의 ICV 영역으로 Aβ를 주입하도록 설계되었습니다. 실험을 시작하기 전에, 대신 Aβ의 파란색 염료와 연습 주사의 예비 기간은 충분한 손재주를 달성하기 위해 장소를해야한다. 즉시 주사 한 결과, 안료 분사가 적절하게 수행되었는지 여부를 확인할 필요가있다. 조심스럽게 뇌를 꺼내 해?...

알츠하이머 병 (AD)의 병리학 적 특징은 그 아밀로이드 β (Aβ)을 감안할 때, AD의 동물 모델의 개발은 Aβ의 신경 과발현에 초점을 맞추고있다. 아밀로이드 전구체 단백질 (APP) 또는 프레 (PS)의 돌연변이는 Aβ 평형 외란 궁극적 가족 성 AD (1)의 발병을 유도하기 때문에, APP 또는 PS 유전자 돌연변이를 포함하는 마우스 모델이 일반적으로 허용되고있다. 형질 전환 마우스의 넓은 범위 중, 원형 마우스 모델은 다음과 같습니다 : TG2576, PDAPP, APP / PS1과 APP23합니다. 뇌에서,이 마우스는 일반적으로 Aβ 응집 결국 노인반을 나타내고; 플라크 형성은 그들이 학습과 기억의 행동 테스트에서 성능 저하를 표시하도록 유의 한인지 ​​장애옵니다. 천연 인간 AD 병리를 모방하는 트랜스 제닉 마우스를 사용하여 생성을 따라서 다이의 진행을 모니터하기 위해 우리시킴으로써 AD 연구 사회에 기여한sea​​se. 이 Aβ 플라크를 개발하기 위해 마우스 개월 취하고 더 긴 Aβ 유도 시냅스 또는 2,3- 행동 이상을 표시하기 때문에, 트랜스 제닉 마우스를 사용하는 것은 비 경제적으로 시간 소모적이다. 원래 형질 전환 마우스 모델의 단점을 극복하기위한 대안으로 개발 된, 비 - 형질 전환 모델은 또한 일반적으로 그들의 독특한 이점 때문에 사용된다. AD와 같은인지 결핍은 다른 물리 화학적으로 유발 될 수있는 반면 병원체 - 유도 AD의 모델 수단 - 예컨대 스코 폴라 민 등의 신경 독성 화합물의 주입, 병변의 유도로, 뇌에 Aβ의 직접 주사에 의해 제조 할 수있다 이러한 해마와 같은 또는 대뇌 피질의 손상 사에 의해 인식 관련 분야이다. 그러나,인지 장애의 비 병원성 유도 정확하게 AD의 기본 병태 생리를 반영하지 않는다; 대신, 그것은 단지 그 결과 증상을 모방. 대조적으로, AD 병원체 - 유도 모델은 A(46), 주사 하였을 마우스 모델은 AD-같은 행동 이상을 표시 할 수 있습니다뿐만 아니라, Aβ 병리, 가족 및 산발적 AD가 공유하는 공통의 기능을 나타낼 수있다. 뇌 조직에서 Aβ 플라크를 시각화하기 어려움에도 불구하고, AD의 조사가 매력적인 만드는 Aβ 주입 모델의 가장 큰 장점은 제어합니다. 연구자들은 마약 관련 연구에서 잘못된 데이터로 이어질 수 마우스 모델의 개별 차이를 걸러 수 있습니다. 적시 약물 치료는 후보 약물의 기전에 따라 활성화되고; 정교한, Aβ 응집 억제제는 Aβ의 주입 전에 적용 할 수있다. 또한, 연구자들은 다른 요소가 긴밀하게 개인차를 포함하여 제어되기 때문에 Aβ 주입 후 발생하는 병원성 변화가 Aβ 노출에서 파생되어 있다고 가정 할 수 있습니다. 시간이 프로토콜에서 생생한 설명흐름은 제시 정위기구없이 Aβ​​의 뇌실 (ICV) 주사를 통해 정상 마우스의 AD-같은 표현형을 유도합니다. 뇌 조직에 주입 - 유발 손상을 최소화하는 구조적 손상 및 병변을 유발 염증 가능성을 방지하기 위해 필수적이다. 마우스 취급 능력의 부족은 뜻밖의 연결 부상으로 이끈다. 또한, 주입하는 동안 달성 될 수있는 적절한 각도 및 깊이를 활성화 기술은 잦은 오류를 회피하는 것이 특히 중요하다. ICV 주사 상세한 선명한 설명에 더하여, 이하의 프로토콜에 의해 생성 된 모델의 신뢰성은 또한 다음 섹션에서 설명된다. 다음 프로토콜함으로써 궁극적으로 AD 사회에 대한 의미있는 발견으로 이어질 수있는 발판을 제공, AD 연구에 기여하는 안정적이고 쉽게 이해 도구​​가 될 수 있습니다.

<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.

이 섹션에서는 Aβ 집계 및 메모리 적자의 Y-미로 평가의 확인에 의해 얻을 수있는 결과의 예를 보여줍니다. 전장 Aβ를 사용하여 42 개의 아미노산 Aβ 모노머, 올리고머의 혼합물, 및 피 브릴 (도 3)의 (1-42) 펩티드를 제조 하였다. HFIP 유도 monomerization 공정을 통해, 상대적으로 균일 한 단량체 (레인 B)를 얻었다. 7 일간 배양 한 후, Aβ 집계 (레인 C)의 다양한 크기를 ...

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

아밀로이드-β (Aβ)의 동물 모델에 정의 된 수량 및 Aβ 단편 종의 관리를 가능하게하고 각 연구 그룹 내의 개별 차이를 줄여 주사 하였을. 이 프로토콜은 정상 마우스에서 알츠하이머와 같은 행동 이상의 생산을 가능하게 정위기구없이 Aβ​​의 뇌실 (ICV) 주입을 설​​명합니다.

일반 마우스의 아밀로이드-β의 펩티드의 뇌 실내 주사는 급성 치매와 같은인지 적자를 유도합니다

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

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

영구와 광범위한 신경 세포의 형질 도입에 대한 신생아 마우스 뇌의 뇌실 바이러스 감염

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

연구

신경과학

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

의 기록 세포 내 전압 응답에 대한 전기 생리 방법<em&gt; 초파리</em&gt; 빛 자극에 광수와의 interneurons<em&gt; 생체</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

전체 셀 전류 두뇌 분할 영역에서 GABA의 퍼프 응용 프로그램에 의해 유도 된

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

수지 성 소와 쥐의 통증 검사를 가진 뇌경치료

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

쥐 모델에서 실험적 지주막하 출혈을 유도하기 위한 자가 혈액의 Pre-chiasmatic, 단일 주사

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

개방형 협업 학습의 뇌 간 싱크로니: fNIRS 하이퍼스캐닝 연구

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 하이퍼스캐닝 연구에서 뇌 간 동기화를 계산하고 검증하는 방법

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

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

고기능 뇌졸중 환자의 인지 결함을 감지하기 위한 이중 작업 스트룹 패러다임

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

생쥐의 운동 피질에서 세포외 전기생리학적 기록

Multichannel Extracellular Recording in Freely Moving Mice

저자 스포트라이트: 자유롭게 움직이는 마우스의 신경 활동을 연구하기 위한 다중 채널 세포외 기록의 발전 

자유롭게 움직이는 마우스의 다채널 세포외 기록

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