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

Denna forskning stöddes av ett bidrag på Korea Health Technology R &amp; D Project via Korea Health Industry Development Institute (KHIDI), som finansieras av ministeriet för hälsa och välfärd, Sydkorea (licensnummer: H14C04660000).

Alla djurförsök utfördes i enlighet med National Institutes of Health Guide för vård och användning av försöksdjur (NIH Publications nr 8023, reviderad 1978) och Animal Care och användning kommittén av Institutional Animal Care och användning kommittén KIST (Seoul, Korea). 1. Animaliska Beredning <ol><li> Förbereda en grupp på 7 veckor gamla ICR-möss (eller C57BL / 6). </li><li> Låt möss gå igenom en acklimatisering process för 3-7 dagar efter transport för att återställa homeostas. I all...

<ol>
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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. M., 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=Correlations+of+amyloid-beta+concentrations+between+CSF+and+plasma+in+acute+Alzheimer+mouse+model.">Correlations of amyloid-beta concentrations between CSF and plasma in acute Alzheimer mouse model.</a> Sci Rep. 4, 6777 (2014).</li></ol>

Det viktigaste steget i detta protokoll är ICV injektion av Ap. Detta protokoll är utformad för att injicera Ap i ICV regionen möss utan stereotaktiska instrument 11,12. Innan du börjar ett experiment, bör en preliminär period av praxis injektioner med ett blått färgämne i stället av Ap äga rum för att uppnå tillräcklig skicklighet. Omedelbart efter injektion är det nödvändigt att kontrollera om pigmentet injektion utfördes på korrekt sätt. Ta försiktigt ut hjärnan och kontrollera om l?...

Med tanke på att amyloid-β (A-beta) är ett patologiskt kännetecken för Alzheimers sjukdom (AD), har utvecklingen av AD djurmodeller fokuserat på neural överuttryck av Ap. Eftersom mutationer i amyloidprekursorprotein (APP) eller presenilin (PS) leda till störningar i Ap jämvikt och slutligen till patogenesen av familjär AD 1, musmodeller involverar APP eller PS genmutationer har varit allmänt accepterat. Bland det stora utbudet av transgena möss, proto musmodeller innefattar följande: TG2576, PDApp, APP / PS1 och APP23. I hjärnan dessa möss uppvisar generellt Ap-aggregation och slutligen senila plack; plackbildning följs av betydande kognitiv nedsättning så att de visar dåliga resultat i beteendetester för inlärning och minne. Genereringen av att använda transgena möss som naturligt efterliknar människo AD patologi har därmed bidragit till AD forskarsamhället genom att tillåta oss att övervaka utvecklingen av dikärlsjukdomar. Men med hjälp av transgena möss är oekonomiskt och tidskrävande eftersom det tar månader för möss att utveckla Ap plack och ännu längre tid att visa Ap-inducerad synaptisk eller beteendeavvikelser 2,3. Ursprungligen utvecklats som ett alternativ för att övervinna bristerna i transgena musmodeller, är icke-transgena modeller används också ofta på grund av deras distinkta fördelar. Patogenfria inducerad AD-modeller kan framställas genom direkt injektion av A-beta in i hjärnan, medan AD-liknande kognitiva brister också kan utlösas av andra kemiska och fysiska medel-såsom injektion av neurotoxiska föreningar såsom skopolamin, induktionen av lesioner i kognitionsrelaterade områden såsom hippocampus, eller genom kortikal skada 4. Men den icke-patogena induktion av kognitiv svikt inte alltid motsvarar den grundläggande patofysiologi AD; istället, bara härmar det de symptomatiska resultat. I motsats till en patogen-inducerad AD-modellen, A46; -inmatade musmodell, kan inte bara visa AD-liknande beteendeavvikelser, men kan också uppvisa Ap patologi, den gemensamma nämnaren delas av familjär och sporadisk AD. Trots att det är svårt att visualisera Ap plack i hjärnvävnaden, är den största fördelen med Ap-injicerade modell som gör det attraktivt för AD undersökning sin styrbarhet. Forskare kan sålla bort de individuella skillnaderna i musmodeller som kan leda till felaktiga uppgifter i narkotikarelaterade studier. Tid läkemedelsbehandling är aktiverad beroende på mekanismen för läkemedelskandidaten; att utarbeta, kan en hämmare av Ap-aggregation tillämpas före injektion av Ap. Dessutom kan utredarna anta att den patogena omvandling som uppstår efter Ap injektionen härrör från Ap exponering eftersom andra faktorer hårt kontrollerad, inklusive individuella skillnader. I detta protokoll, en levande beskrivning av how att framkalla en AD-liknande fenotyp i normala möss via Ap intracerebroventrikulär (ICV) injektion utan stereotaktiska instrument presenteras. Minimera insättnings provocerade skador på hjärnvävnaden är viktigt att förhindra risken för strukturell skada och skada inducerad inflammation. En bristande skicklighet vid hantering mus leder till oväntade neuronal skada. Vidare tekniker som gör det möjligt lämplig vinkel och djup som skall uppnås under injektionen är särskilt viktigt att kringgå ofta misstag. Förutom en detaljerad, levande förklaring av ICV injektion, är tillförlitligheten hos den modell som produceras av följande protokoll också illustreras i de följande avsnitten. Följande protokoll kan vara en tillförlitlig och lättförståeligt verktyg som bidrar till AD forskning, vilket ger en språngbräda som i slutändan kan leda till en meningsfull upptäckt för AD samhället.

<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.

Det här avsnittet visar exempel på resultat som kan erhållas genom bekräftelse av Ap-aggregation och Y-labyrint bedömning av minnesbrister. Med användning av fullängds-A-beta (1-42) peptid av 42 aminosyror, blandning av A-beta-monomerer, oligomerer och fibriller (Figur 3) producerades. Genom HFIP-inducerad monomerise steg, var relativt homogena monomerer (Lane B) erhålls. Efter 7 dagars inkubering, olika storlekar av Ap-aggregat (Lane C) utvecklats. Trimerer och ...

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

Amyloid-β (A-beta) -inmatade djurmodell möjliggör administration av en definierad kvantitet och arter av Ap-fragment och minskar individuella skillnader inom varje studiegrupp. Detta protokoll beskriver intracerebroventrikulära (ICV) injektion av Ap utan stereotaktiska instrument, vilket möjliggör produktion av Alzheimer-liknande beteendeavvikelser i normala möss.

Intracerebroventrikulär Injektion av amyloid-p-peptider hos normala möss att Akut Framkalla Alzheimer-liknande kognitiva brister

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

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

Intracerebroventrikulär Viral Injektion av neonatal mus hjärnans för långlivade och Utbredd neuronal Transduktion

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

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

Elektro Metod för inspelning Intracellulärt Spännings svar av<em&gt; Drosophila</em&gt; Fotoreceptorer och Intern på ljusstimuli<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

Hela-cell strömmar induceras av Puff tillämpningen av GABA i hjärnan skivor

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 och smärtprov hos möss

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, enstaka injektion av autologt blod för att inducera experimentell subaraknoidalblödning i en råttmodell

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-studie

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

Hur man beräknar och validerar synkronisering mellan hjärnor i en fNIRS Hyperscanning-studie

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

Mätning av riktningsinformationsflödet i fNIRS-hyperskanningsdata med hjälp av partialvåglettransformkoherensmetoden

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

Dual-task Stroop Paradigm för att upptäcka kognitiva underskott hos högfungerande strokepatienter

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

Extracellulär elektrofysiologisk registrering i den motoriska cortex hos möss

Multichannel Extracellular Recording in Freely Moving Mice

Författare i fokus: Framsteg inom flerkanalig extracellulär inspelning för att studera neuronal aktivitet hos möss som rör sig fritt 

Flerkanalig extracellulär inspelning i fritt rörliga möss

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