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

Dit onderzoek werd gesteund door een subsidie ​​van de Korea Health Technology R &amp; D Project door de Korea Health Industry Development Institute (KHIDI), gefinancierd door het ministerie van Volksgezondheid en Welzijn, de Republiek Korea (subsidie ​​nummer: H14C04660000).

Alle dierlijke experimenten werden uitgevoerd in overeenstemming met de National Institutes of Health Guide voor de Zorg en gebruik van proefdieren uitgevoerd (NIH Publications No. 8023, herzien 1978) en met de Animal Care en gebruik Comite van de Institutional Animal Care en gebruik Comite van KIST (Seoul, Korea). 1. Dierlijke Voorbereiding <ol><li> Bereid een groep van 7 weken oude mannelijke ICR-muizen (of C57BL / 6). </li><li> Laat de muizen gaan door middel van een acclimatisatie proces voor 3-7 dagen na ...

<ol>
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De belangrijkste stap in dit protocol is de ICV injectie van Ap. Dit protocol is ontworpen om Ap te injecteren in het gebied van ICV muizen zonder stereotactische instrumenten 11,12. Voordat u begint met een experiment, moet een voorafgaande periode van de praktijk injecties met een blauwe kleurstof in plaats van Ap plaatsvinden om voldoende vaardigheid te bereiken. Direct na de injectie moet worden nagegaan of het pigment injectie correct is uitgevoerd. Verwijder voorzichtig de hersenen en controleer of de j...

Aangezien amyloid-β (Aß) is een pathologische kenmerk van de ziekte van Alzheimer (AD) is de ontwikkeling van AD diermodellen gericht op neurale overexpressie van AB. Omdat mutaties in amyloïde precursor proteïne (APP) of preseniline (PS) leiden tot verstoring van AB evenwicht en uiteindelijk tot de pathogenese van familiale AD 1, hebben muismodellen met APP of PS genmutaties algemeen aanvaard. Onder de brede waaier van transgene muizen, prototypische muismodellen onder meer de volgende: Tg2576, PDAPP, APP / PS1 en APP23. In de hersenen, deze muizen vertonen in het algemeen Ap-aggregatie en uiteindelijk seniele plaques; plaquevorming volgt significante cognitieve stoornissen zodat zij vertonen slechte prestaties in gedragstesten van leren en geheugen. Het genereren van transgene muizen die natuurlijk nabootsen menselijke AD pathologie heeft aldus bijgedragen aan AD Onderzoeksvereniging door ons om de progressie van de di bewakenSease. Echter, transgene muizen oneconomisch en tijdrovend omdat het maanden voordat de muizen Ap-plaques en zelfs langer Aß-geïnduceerde synaptische of gedragsafwijkingen 2,3 vertonen. Oorspronkelijk ontwikkeld als alternatief voor de tekortkomingen van transgene muismodellen overwinnen, zijn niet-transgene modellen ook vaak gebruikt vanwege hun verschillende voordelen. Pathogenen geïnduceerde AD modellen kunnen worden geproduceerd door de directe injectie van Ap in de hersenen, terwijl AD-achtige cognitieve tekorten ook worden veroorzaakt door andere chemische en fysische middelen-zoals de injectie van neurotoxische verbindingen zoals scopolamine, de inductie van laesies cognitie-gerelateerde gebieden, zoals de hippocampus, of door het corticale schade 4. Echter, de niet-pathogene inductie van cognitieve stoornissen niet nauwkeurig de fundamentele pathofysiologie van AD; In plaats daarvan bootst alleen de symptomatische resultaten. Daarentegen een door pathogenen geïnduceerde AD model, de A46; -injected muismodel, kan niet alleen laten zien AD-achtige gedragsafwijkingen maar kan ook Ap pathologie, het gemeenschappelijk kenmerk gedeeld door familiale en sporadische AD vertonen. Ondanks de beperkte Ap-plaques te visualiseren in het hersenweefsel, het grootste voordeel van de Aß-geïnjecteerde model dat het aantrekkelijk maakt voor AD onderzoek is controleerbaarheid. Onderzoekers kunnen onkruid uit de individuele verschillen in muismodellen die kunnen leiden tot foutieve gegevens in drugsgerelateerde studies. Tijdige behandeling geneesmiddel wordt geactiveerd afhankelijk van het mechanisme van het testgeneesmiddel; uit te werken, kan een remmer van Ap-aggregatie worden toegepast voor de injectie van Ap. Daarnaast kunnen onderzoekers veronderstellen dat de pathogene transformatie die ontstaat na Aß injectie wordt afgeleid van het Ap belichting omdat andere factoren strak gecontroleerd, met inbegrip van individuele verschillen. In dit protocol, een levendige beschrijving van how met een AD-achtige fenotype in normale muizen via AB intracerebroventriculaire (ICV) injectie te induceren zonder stereotactische instrumenten wordt gepresenteerd. Het minimaliseren inbrengen veroorzaakte schade aan hersenweefsel is essentieel om de mogelijkheid van structurele schade en letsel-geïnduceerde ontsteking te voorkomen. Een gebrek aan vaardigheid in muiswerkzaamheden leidt tot onverwachte neuronale schade. Bovendien technieken die de geschikte hoek en diepte ervan tijdens de injectie te bereiken zijn vooral belangrijk regelmatig fouten omzeilen. Naast een gedetailleerde uitleg van de levendige ICV injectie wordt de betrouwbaarheid van het model door het volgende protocol ook geïllustreerd in de volgende paragrafen. Het volgende protocol kan een betrouwbare en gemakkelijk te begrijpen instrument dat bijdraagt ​​aan AD onderzoek, waardoor een kapstok die uiteindelijk kunnen leiden tot een betekenisvolle ontdekking AD samenleving.

<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.

Dit gedeelte toont voorbeelden van de resultaten die kunnen worden verkregen door de bevestiging van Ap-aggregatie en Y-doolhof beoordeling van tekorten geheugen. Met de volledige lengte Aß (1-42) peptide van 42 aminozuren, mengsel van Ap monomeren, oligomeren en fibrillen (figuur 3) geproduceerd. Door HFIP-geïnduceerde monomerisatie stap werden relatief homogene monomeren (baan B) verkregen. Na 7 dagen incubatie verschillende maten van Ap-aggregaten (baan C) ontwikkel...

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

De amyloid-β (Aß) -injected diermodel kan de toediening van een bepaalde hoeveelheid en de soort van Ap-fragmenten en vermindert de individuele verschillen binnen elke studiegroep. Dit protocol beschrijft de intracerebroventriculaire (ICV) injectie van Ap zonder stereotactische instrumenten, waardoor de productie van Alzheimer-achtige gedragsafwijkingen bij normale muizen.

Intracerebroventriculaire Injectie van amyloid-β peptiden in normale muizen om acuut induceren Alzheimer-achtige cognitieve tekorten

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

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

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