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

Denne forskningen ble støttet av en bevilgning av Korea Health Technology R &amp; D-prosjektet gjennom Korea Health Industry Development Institute (KHIDI), finansiert av Helsedepartementet og velferd, Republikken Korea (tilskudd nummer: H14C04660000).

Alle dyreforsøk ble utført i samsvar med National Institutes of Health Guide for omsorg og bruk av forsøksdyr (NIH publikasjoner nr 8023, revidert 1978) og med Animal Care og bruk komité Institutional Animal Care og bruk komité Kist (Seoul, Korea). 1. Animal Forberedelse <ol><li> Forberede en gruppe på syv uker gamle ICR hannmus (eller C57BL / 6). </li><li> La musene går gjennom en akklimatiserings fremgangsmåte for 3-7 dager etter transport for å gjenopprette homeostase. Generelt, plasserer en gruppe...

<ol>
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Det viktigste i denne protokollen er ICV injeksjon av Ap. Denne protokollen er utformet for å injisere Ap inn i ICV region av mus uten stereotaktiske instrumenter 11,12. Før et eksperiment, må en innledende periode med praksis injeksjoner med et blått farvestoff i stedet for Ap finne sted for å oppnå tilstrekkelig bevegelighet. Umiddelbart etter injeksjonen, er det nødvendig å kontrollere om pigmentet injeksjonen ble utført riktig. Forsiktig ta ut hjernen og sjekke om den aktuelle regionen ble farget...

Gitt at amyloid-β (Ap) er en patologisk kjennetegn på Alzheimers sykdom (AD), har utviklingen av AD dyremodeller fokusert på nevrale overekspresjon av Ap. Ettersom mutasjoner i amyloid forløper protein (APP) eller presenilin (PS) føre til forstyrrelse av Ap likevekt og til slutt til patogenesen av familiær AD 1, har musemodeller som omfatter APP eller PS-genmutasjoner blitt generelt akseptert. Blant det store utvalget av transgene mus, prototypiske musemodeller inkluderer følgende: TG2576, PDAPP, APP / PS1 og APP23. I hjernen, disse mus oppviser generelt Ap aggregering og til slutt senile plakk; plakkdannelse er etterfulgt av en betydelig kognitiv svikt, slik at de viser dårlig ytelse i atferdstester for læring og hukommelse. Den generasjonen av å bruke transgene mus som naturlig etterligne menneskelig AD patologi har dermed bidratt til AD forskning samfunnet ved å tillate oss å overvåke utviklingen av diSease. Imidlertid, ved bruk av transgene mus er uøkonomisk og tidkrevende fordi det tar måneder for mus å utvikle Ap-plakk, og enda lenger for å vise Ap-induserte synaptiske eller unormal oppførsel 2,3. Opprinnelig utviklet som et alternativ til å overvinne svakhetene i transgene musemodeller, er ikke-transgene modeller også ofte brukt på grunn av deres distinkte fordeler. Patogen-indusert AD-modeller kan fremstilles ved direkte injeksjon av Ap i hjernen, mens AD-lignende kognitive mangler kan også utløses av andre kjemiske og fysikalske midler, for eksempel injeksjon av nevrotoksiske forbindelser slik som scopolamin, induksjon av lesjoner i kognisjon relaterte områder som hippocampus, eller ved kortikal skade fire. Men den ikke-patogene induksjon av kognitiv svikt ikke nøyaktig gjenspeiler fundamentale patofysiologien av AD; i stedet, det bare etterligner sine symptomatiske resultater. I motsetning til dette, et patogen-indusert AD modellen, A46; -injected musemodell, kan ikke bare vise AD-lignende unormal oppførsel, men kan også vise Ap patologi, den felles trekk deles av familiær og sporadisk AD. Til tross for vanskeligheten med å visualisere Ap-plakk i hjernevevet, er den største fordelen med Ap-injiserte modell som gjør det attraktivt for AD undersøkelse dens kontrollerbarhet. Forskere kan luke ut de individuelle forskjellene i musemodeller som kan føre til feilaktige data i narkotikarelaterte studier. Betimelig medikamentbehandling er aktivert avhengig av mekanismen av legemiddelkandidat; å utdype, kan en inhibitor av Ap aggregering påføres før injeksjonen av Ap. I tillegg kan undersøkere anta at den patogene transformasjon som oppstår etter injeksjon Ap er avledet fra Ap eksponering fordi de andre faktorene er strengt kontrollert, inkludert individuelle forskjeller. I denne protokollen, en levende beskrivelse av how å indusere en AD-lignende fenotype i normale mus via Ap intracerebroventrikulær (ICV) injeksjon uten stereotaktiske instrumenter er presentert. Minimalisering av innsettings-fremkalt skade på hjernevevet er avgjørende for å hindre muligheten for strukturelle skader og lesjon-indusert inflammasjon. En mangel på dyktighet i mus håndtering fører til uventede neuronal skade. Videre teknikker som muliggjør passende vinkel og dybde som skal oppnås i løpet av injeksjonen er spesielt viktig for å omgå hyppige feil. I tillegg til et detaljert, levende forklaring av ICV injeksjon, er påliteligheten av modellen produsert av følgende protokoll også illustrert i følgende avsnitt. Den følgende protokollen kan være en pålitelig og lett forståelig verktøy som bidrar til AD forskning, for derved å tilveiebringe en byggestein som kan til slutt føre til en meningsfylt oppdagelse for AD samfunnet.

<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.

Denne delen viser eksempler på resultater som kan oppnås ved bekreftelse av Ap aggregering og Y-labyrint vurdering av hukommelsessvikt. Ved hjelp av full-lengde Ap (1-42) peptid med 42 aminosyrer, blanding av Ap-monomerer, oligomerer, og fibriler (figur 3) ble fremstilt. Gjennom HFIP-indusert monomerization trinnet, ble relativt homogene monomerer (Lane B) oppnådd. Etter 7 dagers inkubasjonstid, ulike størrelser av Ap-aggregater (Lane C) utviklet. Trimerer og tetrame...

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

Den amyloid-β (Ap) -injected dyremodell gjør administrasjonen av en definert mengde og arter av Ap-fragmenter og reduserer individuelle forskjeller innen hver studie gruppe. Denne protokollen beskriver intracerebroventrikulær (ICV) injeksjon av Ap uten stereotaktiske instrumenter, slik at produksjon av abnormiteter Alzheimer-lignende adferds hos normale mus.

Intracerebroventrikulær Injeksjon av amyloid-beta Peptider i normale mus til Akutt Fremkall 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, ...

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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Hele celle strøm av Puff søknad GABA i hjernen skiver

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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 ...

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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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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

Hvordan beregne og validere inter-hjernesynkronisering i en fNIRS Hyperscanning Study

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

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

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Multichannel Extracellular Recording in Freely Moving Mice

Forfatter Spotlight: Fremskritt innen flerkanals ekstracellulær opptak for å studere nevronal aktivitet hos mus som beveger seg fritt 

Flerkanals ekstracellulært opptak i fritt bevegelige mus

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