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 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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Y., 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=Taurine+in+drinking+water+recovers+learning+and+memory+in+the+adult+APP/PS1+mouse+model+of+Alzheimer's+disease.">Taurine in drinking water recovers learning and memory in the adult APP/PS1 mouse model of Alzheimer's disease.</a> Sci Rep. 4, 7467 (2014).</li><li>Paxinos, G., Franklin, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. 2nd edn. , (2001).</li><li>Ledo, J. 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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-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) -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, ...

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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Intracerebroventrikulær Viral Injektion af Neonatal Mouse Hjerne for Vedvarende og Udbredt Neuronal transduktion

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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

Elektrofysiologiske Metode til Recording Intracellulær Voltage Responses af<em&gt; Drosophila</em&gt; Fotoreceptorer og interneuroner til Light Stimuli<em&gt; In vivo</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

Hele-celle strømninger induceret af Puff anvendelse af GABA i hjernen skiver

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

Intracerebroventricular behandling med resiniferatoxin og smertetest hos mus

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

Pre-chiasmatisk, enkelt injektion af autologt blod for at inducere eksperimentel subaraknoid blødning i en rottemodel

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

Inter-Brain Synchrony i Open-Ended Collaborative Learning: En fNIRS-Hyperscanning Study

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

Sådan beregnes og valideres synkronisering mellem hjerner i en fNIRS Hyperscanning-undersøgelse

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

Måling af retningsbestemt informationsstrøm i fNIRS-hyperscanningsdata ved hjælp af metoden Delvis 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

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

Ekstracellulær elektrofysiologisk optagelse i den motoriske cortex hos mus

Multichannel Extracellular Recording in Freely Moving Mice

Forfatter Spotlight: Fremskridt inden for multikanals ekstracellulær optagelse til undersøgelse af neuronal aktivitet hos frit bevægelige mus 

Multikanals ekstracellulær optagelse i frit bevægelige 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 ...