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-ÃŽÂ² Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits at JoVE.com

Denne forskning blev støttet af en bevilling på Korea Health Technology R &amp; D Project via Korea Health Industry Development Institute (KHIDI), finansieret af Ministeriet for Sundhed &amp; Velfærd, Republikken Korea (tilskud nummer: H14C04660000).

Alle eksperimenter dyr blev udført i overensstemmelse med National Institutes of Health Guide til Pleje og anvendelse af forsøgsdyr (NIH Publications nr 8023, revideret 1978) og med Animal Care og brug Udvalg for Institutional Animal Care og brug Udvalg KIST (Seoul, Korea). 1. Dyrepræparation <ol><li> Forbered en gruppe af 7 uger gamle ICR-mus (eller C57BL / 6). </li><li> Lad musene gå gennem et akklimatisering proces i 3-7 dage efter transport at genoprette homeostase. Generelt placere en gruppe på 4-5 m...

<ol>
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Det vigtigste skridt i denne protokol er ICV injektion af Ap. Denne protokol er udviklet til at injicere Ap ind ICV-regionen af mus uden stereotaktiske instrumenter 11,12. Før du starter et eksperiment, bør en foreløbig periode med praksis injektioner med et blåt farvestof i stedet for Ap finde sted for at opnå tilstrækkelig fingerfærdighed. Umiddelbart efter injektionen, er det nødvendigt at kontrollere, om pigmentet injektion blev udført korrekt. Forsigtigt tage hjernen og kontrollere, om det relev...

Da amyloid-β (Ap) er en patologisk kendetegn for Alzheimers sygdom (AD), har udviklingen af ​​AD dyremodeller fokuseret på neurale overekspression af Ap. Fordi mutationer i amyloid precursor protein (APP) eller presenilin (PS) føre til forstyrrelse af Ap ligevægt og i sidste ende til patogenesen af familiær AD 1, har musemodeller involverer APP eller PS genmutationer blevet almindeligt accepteret. Blandt den brede vifte af transgene mus, prototypiske musemodeller omfatter følgende: Tg2576, PDAPP, APP / PS1 og APP23. I hjernen disse mus udviser generelt Ap aggregation og vinder senile plaques; plakdannelse efterfølges af betydelig kognitiv svækkelse, således at de viser dårlige resultater i adfærdsmæssige tests af indlæring og hukommelse. Frembringelsen af ​​at anvende transgene mus, der naturligt efterligne menneskelige AD patologi har dermed bidraget til AD forskning samfund ved at lade os overvåge progression af disease. Men ved hjælp transgene mus er uøkonomisk og tidskrævende, fordi det tager måneder for mus for at udvikle Ap plaques og endnu længere tid at vise Ap-induceret synaptic eller adfærdsmæssige abnormiteter 2,3. Oprindeligt udviklet som et alternativ til at overvinde manglerne i transgene musemodeller, er ikke-transgene modeller også almindeligt anvendt på grund af deres forskellige fordele. Patogen-induceret AD modeller kan fremstilles ved direkte injektion af Ap i hjernen, hvorimod AD-lignende kognitive mangler også kan udløses af andre kemiske og fysiske midler-såsom injektion af neurotoksiske forbindelser, såsom scopolamin, induktionen af ​​læsioner i kognition-relaterede områder som hippocampus, eller ved kortikal skade 4. Men den ikke-patogene induktion af kognitiv svækkelse ikke præcist afspejler grundlæggende patofysiologi af AD; I stedet er det kun efterligner sine symptomatiske resultater. Derimod kan et patogen-induceret AD modellen, A46; -injiceret musemodel, kan ikke kun vise AD-lignende adfærdsmæssige abnormiteter, men kan også udvise Ap patologi, det fælles træk deles af familiær og sporadisk AD. Trods problemerne med at visualisere Ap plaques i hjernevævet, den største fordel for Ap-injicerede model, der gør det attraktivt for AD undersøgelse er dens styrbarhed. Forskere kan frasortere de individuelle forskelle i musemodeller, der kan føre til fejlagtige data i narkotikarelaterede undersøgelser. Rettidig lægemiddelbehandling er aktiveret afhængigt af mekanismen af ​​kandidat narkotika; at udarbejde, kan en inhibitor af AB sammenlægning anvendes før injektionen af ​​Ap. Derudover kan undersøgere antage, at den patogene transformation, der opstår efter Ap injektion er afledt af Ap eksponering fordi de andre faktorer er tæt kontrolleret, herunder individuelle forskelle. I denne protokol, en levende beskrivelse af how at fremkalde en AD-lignende fænotype i normale mus via Ap intracerebroventrikulær (ICV) injektion uden stereotaktiske instrumenter præsenteres. Minimering indsættelse-provokeret skader på hjernevævet er afgørende for at forhindre muligheden for strukturelle skader og læsion-induceret inflammation. En manglende evner i muse håndtering medfører uventet neuronal skade. Endvidere teknikker, der muliggør en passende vinkel og dybde, der skal opnås under injektionen er særligt vigtigt at omgå hyppige fejl. Foruden en detaljeret, levende forklaring af ICV injektion pålideligheden af ​​modellen produceret af den følgende protokol også illustreret i de følgende afsnit. Følgende protokol kan være en pålidelig og let forståelig værktøj, der bidrager til AD forskning, hvilket giver en springbræt, der i sidste ende kan føre til en meningsfuld opdagelse for AD samfund.

<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.

Dette afsnit viser eksempler på de resultater, der kan opnås ved bekræftelse af Ap-aggregering og Y-labyrint vurdering af hukommelsessvigt. Brug af fuld længde Ap (1-42) peptid af 42 aminosyrer, blanding af Ap-monomerer, oligomerer og fibriller (figur 3) blev produceret. Gennem HFIP-induceret monomerisering trin blev relativt ensartede monomerer (bane B) opnået. Efter de 7 dages inkubation, diverse størrelser af Ap aggregater (bane C) udviklede. Trimerer og tetrame...

Watch this Scientific Journal Video about Intracerebroventricular Injection of Amyloid-ÃŽÂ² Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits at JoVE.com

Intracerebroventricular Injection of Amyloid-ÃƒÅ½Ã‚Â² Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Den amyloid-β (Ap) -injiceret dyremodel muliggør administration af en defineret mængde og art af Ap fragmenter og reducerer individuelle forskelle inden for hver studiegruppe. Denne protokol beskriver intracerebroventrikulær (ICV) injektion af Ap uden stereotaktiske instrumenter, muliggør produktionen af ​​Alzheimer-lignende adfærdsmæssige abnormiteter i normale mus.

Intracerebroventrikulær Injektion af amyloid-p-peptider i normale mus til Akut Fremkald Alzheimer-lignende kognitive mangler

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

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

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