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

Questa ricerca è stata sostenuta da una sovvenzione della Corea del Health Technology R &amp; S del progetto attraverso la Corea Health Industry Development Institute (Khidi), finanziato dal Ministero della Salute e del Welfare, Repubblica di Corea (codice di autorizzazione: H14C04660000).

Tutti gli esperimenti sugli animali sono stati eseguiti in conformità con il National Institutes of Health Guide per la cura e l&#39;uso di animali da laboratorio (NIH Pubblicazioni n ° 8023, riveduta 1978) e con il Comitato cura e l&#39;uso degli animali del Comitato Cura e uso istituzionale degli animali di KIST (Seoul, Corea). 1. Preparazione degli animali <ol><li> Preparare un gruppo di 7 settimane di età topi ICR maschi (o C57BL / 6). </li><li> Lasciate che i topi passare attraverso un processo di accl...

<ol>
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V., 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=Amelioration+of+Alzheimer's+disease+by+neuroprotective+effect+of+sulforaphane+in+animal+model.">Amelioration of Alzheimer's disease by neuroprotective effect of sulforaphane in animal model.</a> Amyloid. 20, 7-12 (2013).</li><li>Jackson, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VTE+on+an+elevated+T-maze.">VTE on an elevated T-maze.</a> J Comp Psychol. 36, 9 (1943).</li><li>Kim, H. 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. H., 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=Amyloid-beta+oligomers+link+depressive-like+behavior+and+cognitive+deficits+in+mice.">Amyloid-beta oligomers link depressive-like behavior and cognitive deficits in mice.</a> Mol Psychiatry. 18, 1053-1054 (2013).</li><li>Laursen, S. E., Belknap, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebroventricular+injections+in+mice.+Some+methodological+refinements.">Intracerebroventricular injections in mice. 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P., 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=Memantine+rescues+transient+cognitive+impairment+caused+by+high-molecular-weight+abeta+oligomers+but+not+the+persistent+impairment+induced+by+low-molecular-weight+oligomers.">Memantine rescues transient cognitive impairment caused by high-molecular-weight abeta oligomers but not the persistent impairment induced by low-molecular-weight oligomers.</a> J Neurosci. 33, 9626-9634 (2013).</li><li>Haley, T. J., McCormick, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+effects+produced+by+intracerebral+injection+of+drugs+in+the+conscious+mouse.">Pharmacological effects produced by intracerebral injection of drugs in the conscious mouse.</a> Br J Pharmacol Chemother. 12, 12-15 (1957).</li><li>Harkany, T., 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=Neuroprotective+approaches+in+experimental+models+of+beta-amyloid+neurotoxicity:+relevance+to+Alzheimer's+disease.">Neuroprotective approaches in experimental models of beta-amyloid neurotoxicity: relevance to Alzheimer's disease.</a> Prog Neuropsychopharmacol Biol Psychiatry. 23, 963-1008 (1999).</li><li>Wang, D., 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=The+allosteric+potentiation+of+nicotinic+acetylcholine+receptors+by+galantamine+ameliorates+the+cognitive+dysfunction+in+beta+amyloid25-35+i.c.v.-injected+mice:+involvement+of+dopaminergic+systems.">The allosteric potentiation of nicotinic acetylcholine receptors by galantamine ameliorates the cognitive dysfunction in beta amyloid25-35 i.c.v.-injected mice: involvement of dopaminergic systems.</a> Neuropsychopharmacology. 32, 1261-1271 (2007).</li><li>Yamada, K., Nabeshima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+Alzheimer's+disease+and+evaluation+of+anti-dementia+drugs.">Animal models of Alzheimer's disease and evaluation of anti-dementia drugs.</a> Pharmacol Ther. 88, 93-113 (2000).</li><li>Cho, S. 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Il passo più importante in questo protocollo è l&#39;iniezione ICV di Ap. Questo protocollo è progettato per iniettare Ap nella regione ICV di topi senza strumenti stereotassica 11,12. Prima di iniziare un esperimento, un periodo preliminare di iniezioni di pratica con un colorante blu invece di Ap dovrebbe avvenire per raggiungere destrezza sufficiente. Subito dopo l&#39;iniezione, è necessario verificare se l&#39;iniezione di pigmento è stato eseguito correttamente. Estrarre con attenzione il cervello ...

Dato che amiloide-β (Ap) è una caratteristica patologica della malattia di Alzheimer (AD), lo sviluppo di modelli animali di AD si è concentrata sulla sovraespressione neurale di Ap. Poiché le mutazioni nella proteina precursore dell&#39;amiloide (APP) o presenilina (PS) portano a disturbi di Ap equilibrio e, infine, alla patogenesi di AD familiare 1, modelli murini che coinvolgono APP o PS mutazioni del gene sono state generalmente accettata. Tra la vasta gamma di topi transgenici, modelli prototipali di topo sono i seguenti: Tg2576, PDAPP, APP / PS1 e APP23. Nel cervello, questi topi in genere mostrano aggregazione Ap e placche senili alla fine; formazione di placca è seguito da un significativo deterioramento cognitivo tale che essi mostrano scarse prestazioni nei test comportamentali di apprendimento e memoria. La generazione di utilizzare topi transgenici che imitano naturalmente patologia AD umano ha così contribuito alla società di ricerca dC da permettendoci di monitorare la progressione della diSease. Tuttavia, utilizzando topi transgenici è antieconomica e richiede molto tempo perché ci vogliono mesi per i topi di sviluppare placche Ap e anche di più per mostrare sinaptica Ap-indotto o anomalie comportamentali 2,3. Originariamente sviluppato come alternativa per superare le carenze di modelli di topi transgenici, modelli non transgenici sono comunemente utilizzati a causa della loro vantaggi. modelli AD patogeno-indotta possono essere ottenuti mediante iniezione diretta di Ap nel cervello, che deficit cognitivi AD-simili possono anche essere attivati ​​da altri chimici e fisici mezzi, ad esempio l&#39;iniezione di composti neurotossici come scopolamina, l&#39;induzione di lesioni nelle zone cognizione legati, come l&#39;ippocampo, o da danni corticale 4. Tuttavia, l&#39;induzione non patogeno di deterioramento cognitivo non riflette accuratamente la fisiopatologia fondamentale di AD; invece, imita solo i suoi risultati sintomatici. Al contrario, un modello AD patogeno-indotta, l&#39;A46; il modello -injected del mouse, può non solo mostrare anomalie comportamentali AD-like, ma può anche esporre patologia Ap, la caratteristica comune condiviso da AD familiare e sporadica. Nonostante la difficoltà di visualizzare placche Ap nel tessuto cerebrale, il più grande vantaggio del modello di Ap-iniettato che lo rende attraente per le indagini AD è la sua controllabilità. I ricercatori possono estirpare le differenze individuali nel modelli murini che possono portare a dati erronei in studi legati alla droga. trattamento farmacologico attuale è abilitato a seconda del meccanismo del farmaco candidato; di elaborare, un inibitore di Ap aggregazione può essere applicato prima l&#39;iniezione di Ap. Inoltre, i ricercatori possono assumere che la trasformazione patogeno che sorge dopo l&#39;iniezione Ap deriva dall&#39;esposizione Ap perché gli altri fattori sono strettamente controllate, compresa differenze individuali. In questo protocollo, una vivida descrizione di how per indurre un fenotipo AD-come in topi normali via Ap intracerebroventricolare (ICV) di iniezione senza strumenti stereotassica è presentato. Riducendo al minimo i danni inserimento-provocati al tessuto cerebrale è essenziale per evitare la possibilità di danni strutturali e infiammazione lesione indotta. La mancanza di abilità nel maneggiare il mouse porta a danno neuronale inaspettato. Inoltre, le tecniche che consentono l&#39;inclinazione e profondità appropriata da raggiungere durante l&#39;iniezione sono particolarmente importanti per aggirare errori frequenti. Oltre ad una dettagliata spiegazione vivido della iniezione ICV, l&#39;affidabilità del modello prodotto dalla seguente protocollo è anche illustrata nelle seguenti sezioni. Il seguente protocollo potrebbe essere uno strumento affidabile e di facile comprensione che contribuisce alla ricerca dC, fornendo in tal modo un trampolino di lancio che potrebbe in ultima analisi portare ad una scoperta significativa per la società AD.

<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.

Questa sezione illustra esempi dei risultati ottenibili da una conferma di aggregazione Ap e valutazione Y-labirinto di deficit di memoria. Utilizzando il full-length Ap (1-42) peptide di 42 aminoacidi, miscela di monomeri, oligomeri, Ap e fibrille (Figura 3) è stato prodotto. Attraverso il passo monomerization HFIP indotta, sono stati ottenuti monomeri relativamente omogenee (Corsia B). Dopo l&#39;incubazione di 7 giorni, diversi formati di aggregati Ap (corsia C) svil...

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

L&#39;amiloide-β (Ap) -injected modello animale consente la somministrazione di una quantità e specie di frammenti Ap definiti e riduce le differenze individuali all&#39;interno di ciascun gruppo di studio. Questo protocollo descrive la intracerebroventricolare (ICV) iniezione di Ap senza strumenti stereotassica, consentendo la produzione di anomalie comportamentali Alzheimer-like nei topi normali.

Iniezione intracerebroventricular dei peptidi beta-amiloide in topi normali per indurre Acutamente Alzheimer-like Deficit cognitivi

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.

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Autore in primo piano: Progressi nella registrazione extracellulare multicanale per lo studio dell'attività neuronale nei topi che si muovono liberamente 

Registrazione extracellulare multicanale in topi che si muovono liberamente

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