Name: quantitative 3d in silico modeling (q3dism) of cerebral amyloid-beta phagocytosis in rodent models of alzheimer's disease
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Description: Watch this Scientific Journal Video about Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease at JoVE.com

M-V.G-S. is supported by a BrightFocus Foundation Alzheimer's Disease Research Fellowship Award (A2015309F) and an Alzheimer's Association, California Southland Chapter Young Investigator Award. T.M.W. is supported by an ARCS Foundation and John Douglas French Alzheimer's Foundation Maggie McKnight Russell-JDFAF Memorial Postdoctoral Fellowship. This work was supported by the National Institute on Neurologic Disorders and Stroke (1R01NS076794-01, to T.T.), an Alzheimer's Association Zenith Fellows Award (ZEN-10-174633, to T.T.), and an American Federation of Aging Research/Ellison Medical Foundation Julie Martin Mid-Career Award in Aging Research (M11472, to T.T.). We are grateful for startup funds from the Zilkha Neurogenetic Institute, which helped to make this work possible. &#8195;

Dichiarazione di etica della ricerca: Tutti gli esperimenti che coinvolgono animali descritti nel presente documento sono stati approvati dalla University of Southern California Istituzionale cura degli animali e del Comitato uso (IACUC) ed eseguito in stretta conformità con National Institutes of linee guida per la salute e le raccomandazioni della Associazione per la valutazione e l&#39;accreditamento del laboratorio Animal Care International. 1. roditori Cervello Isolamento e preparazione per ...

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Il protocollo che descriviamo in questa relazione per una vera quantificazione 3D di Ap fagocitosi in vivo dai fagociti mononucleari si basa sulla etichettatura specifica dei compartimenti cellulari e subcellulari così come depositi di Ap. In particolare, usiamo Iba1 (ionizzata-calcio Binding Adaptor molecola 1), una proteina che è coinvolta nella arruffarsi membrana e fagocitosi delle cellule dopo l&#39;attivazione 18, 19, a macchia fagociti mononucleari cerebrali. Mentre le ...

La malattia di Alzheimer (AD), la demenza legata all&#39;età più comune 1, è caratterizzata da accumulo cerebrale amiloide-β (Ap) come placche &quot;senili&quot; beta-amiloide, neuroinfiammazione cronica di basso livello, tauopathy, perdita neuronale, e disturbi cognitivi 2 . In AD cervelli di pazienti, neuroinflammation è destinato dagli astrociti e fagociti mononucleari reattiva (indicato come microglia, anche se le loro centrali vs. origine periferica rimane poco chiaro) che circonda depositi Ap 3. Come le sentinelle del sistema immunitario innato del sistema nervoso centrale, microglia sono posizionati centralmente per cancellare il cervello Ap. Tuttavia, il reclutamento della microglia placche Ap è accompagnata da molto piccolo, all&#39;occorrenza, Ap fagocitosi 4,5. Una ipotesi è che la microglia sono inizialmente neuroprotettivi da phagocytozing piccole assemblee di Ap. Tuttavia, alla fine queste cellule diventano neurotossico come opprimente fardello Ap e / o legate all&#39;età d funzionaleecline, provoca microglia in un fenotipo proinfiammatoria disfunzionale, contribuendo alla neurotossicità e cognitivo declino 6. Recenti studi genome-wide (GWAS Association) hanno identificato un gruppo di alleli di rischio AD appartenenti al nucleo innate percorsi immunitario 7 che modulano la fagocitosi 8-11. Di conseguenza, la risposta immunitaria alla deposizione di amiloide cerebrale è diventato un importante area di interesse, sia in termini di comprensione AD eziologia e per lo sviluppo di nuovi approcci terapeutici 12-14. Tuttavia, vi è la necessità vitale per metodologia per valutare fagocitosi Ap in vivo. Per rispondere a questa necessità insoddisfatte, abbiamo sviluppato 3D quantitativa in silico di modellazione (q3DISM) per consentire una vera quantificazione 3D cerebrale fagocitosi Ap dai fagociti mononucleari in modelli di roditori della malattia di Alzheimer-like. Limitata solo dalla misura in cui esse ricapitolare malattia, modelli animali hannodimostrato prezioso per la comprensione AD pathoetiology e per la valutazione delle terapie sperimentali. A causa del fatto che le mutazioni nei geni presenilina (PS) e della proteina precursore dell&#39;amiloide (APP) causano indipendentemente autosomica dominante dC, questi transgeni mutanti sono stati ampiamente utilizzati per generare modelli di roditori transgenici. Topi transgenici APP / PS1 coexpressing simultaneamente &quot;svedese&quot; mutante umano APP (APP SWE) e Δ esone 9 mutante presenilina umana 1 (PS1ΔE9) presente con accelerata amiloidosi cerebrale e neuroinfiammazione 15,16. Inoltre, abbiamo generato topi bi-transgenici coinjected con APP SWE e costrutti PS1ΔE9 (linea TgF344-AD, su uno sfondo di Fischer 344). A differenza dei modelli di topi transgenici di amiloidosi cerebrale, ratti TgF344-AD sviluppano amiloide cerebrale che precede tauopathy, perdita apoptotica di neuroni, e compromissione del comportamento 17. In questo rapporto, si descrive un protocollo per immunostaining microglia, phagolysosomes e depositi Ap in sezioni del cervello di topi APP / PS1 e ratti TgF344-AD, e l&#39;acquisizione di immagini di grandi dimensioni confocale z-dimensionale. Noi dettaglio nella generazione e l&#39;analisi di veri ricostruzioni 3D di set di dati confocale che consentono la quantificazione di Ap assorbimento in phagolysosomes microgliali silico. Più in generale, la metodologia che we dettaglio qui può essere utilizzato per quantificare praticamente qualsiasi forma di fagocitosi in vivo.

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			<td height="40" style="height:40px;width:384px;">Isoflurane</td>
			<td style="width:152px;">Abbott</td>
			<td style="width:127px;">NDC 0044-5260-05</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Dissecting scissors</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">82027-582</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Dissecting scissors Blunt tip</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">82027-588</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Tweezers</td>
			<td style="width:152px;">VWR</td>
			<td>94024-408</td>
			<td></td>
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			<td height="20" style="height:20px;width:384px;">23G needle</td>
			<td style="width:152px;">VWR</td>
			<td>BD305145</td>
			<td></td>
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			<td height="20" style="height:20px;width:384px;">peristaltic pump FH10</td>
			<td style="width:152px;">Thermo Scientific</td>
			<td>72-310-010</td>
			<td></td>
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			<td height="20" style="height:20px;width:384px;">PBS 10X</td>
			<td style="width:152px;">Bioland Scientific</td>
			<td style="width:127px;">PBS01-02</td>
			<td>Working concentration 1X</td>
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			<td height="20" style="height:20px;width:384px;">Adult Mouse Brain Matrix, Coronal slices, Stainless Steel 1mm&#160;</td>
			<td style="width:152px;">Kent Scientific</td>
			<td>RBMS-200C</td>
			<td></td>
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			<td height="20" style="height:20px;width:384px;">Adult Rat Brain Matrix, Coronal slices, Stainless Steel 1mm&#160;</td>
			<td style="width:152px;">Kent Scientific</td>
			<td>RBMS-305C&#160;</td>
			<td></td>
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			<td height="20" style="height:20px;width:384px;">32% Paraformaldehyde aqueous solution</td>
			<td style="width:152px;">EMS</td>
			<td style="width:127px;">15714-S</td>
			<td>Caution: Toxic. Working concentration 4% in PBS</td>
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			<td height="20" style="height:20px;width:384px;">Ethanol</td>
			<td style="width:152px;">VWR</td>
			<td>89125-188</td>
			<td>Various concentrations, see protocol</td>
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			<td height="20" style="height:20px;width:384px;">Tissue-Tek Uni-cassettes Sakura</td>
			<td style="width:152px;">VWR</td>
			<td>25608-774</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Embedding and Infiltration Paraffin</td>
			<td style="width:152px;">VWR</td>
			<td>15147-839</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Microtome Leica RM2125</td>
			<td style="width:152px;">Leica Biosystems</td>
			<td style="width:127px;"></td>
			<td></td>
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			<td height="20" style="height:20px;width:384px;">Disposable Microtome Blades&#160;</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">25608-964</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Water bath Leica HI 1210</td>
			<td style="width:152px;">Leica Biosystems</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Micro slide Superfrost plus</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">48311-703</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Xylene</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:127px;">534056-4X4L</td>
			<td style="width:249px;">Caution: Toxic&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Target Retrieval Solution 10X</td>
			<td style="width:152px;">DAKO</td>
			<td style="width:127px;">S1699</td>
			<td>Working concentration 1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KimWipes</td>
			<td style="width:152px;">VWR</td>
			<td>21905-026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Hydrophobic PAP pen</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">95025-252</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Triton X-100</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">97062-208</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Normal Donkey Serum</td>
			<td style="width:152px;">Jackson Immuno</td>
			<td style="width:127px;">017-000-121</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Coverslips</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">48393081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Prolong Gold antifade reagent with DAPI</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:127px;">P36935</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Glass Slide Rack</td>
			<td style="width:152px;">VWR</td>
			<td style="width:127px;">100492-942</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Iba1 antibody (polyclonal, rabbit)</td>
			<td style="width:152px;">Wako</td>
			<td style="width:127px;">019-19741&#160;</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Iba1 antibody (polyclonal, goat)</td>
			<td style="width:152px;">LifeSpan Bioscience</td>
			<td style="width:127px;">LS-B2645</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">rat CD68 [KP1] antibody (monoclonal, mouse)</td>
			<td style="width:152px;">Abcam</td>
			<td style="width:127px;">ab955</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">mouse CD68 [FA-11] antibody (monoclonal, rat)</td>
			<td style="width:152px;">Abcam</td>
			<td style="width:127px;">ab53444</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">mouse CD107a (LAMP1) antibody (monoclonal, rat)</td>
			<td style="width:152px;">Affymetrix</td>
			<td style="width:127px;">14-1071</td>
			<td>Working concentration 1:100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Beta-Amyloid, 17-24 (4G8) antibody (monoclonal, mouse)</td>
			<td style="width:152px;">Covance</td>
			<td style="width:127px;">SIG-39220</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Beta-Amyloid, 1-16 (6E10) antibody (monoclonal, mouse)</td>
			<td style="width:152px;">Covance</td>
			<td style="width:127px;">SIG-39320</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">OC antibody (polyclonal, rabbit)</td>
			<td colspan="2">Gifted by D. H. Cribbs and C. G. Glabe (UC Irvine)</td>
			<td>Working concentration 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Alexa Fluor 488&#160; mouse secondary antibody</td>
			<td style="width:152px;">Invitrogen</td>
			<td style="width:127px;">A-11001</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Alexa Fluor 488&#160; rat secondary antibody</td>
			<td style="width:152px;">Invitrogen</td>
			<td>A-11006</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Alexa Fluor 594 rabbit secondary antibody</td>
			<td style="width:152px;">Invitrogen</td>
			<td>A-11037</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Alexa Fluor 594 goat secondary antibody</td>
			<td style="width:152px;">Invitrogen</td>
			<td>A-11080</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Alexa Fluor 647 mouse secondary antibody</td>
			<td style="width:152px;">Invitrogen</td>
			<td>A-21235</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Alexa Fluor 647 rabbit secondary antibody</td>
			<td style="width:152px;">Invitrogen</td>
			<td>A-21443</td>
			<td>Working concentration 1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Immersion oil</td>
			<td style="width:152px;">Nikon&#160;</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">A1 Confocal microscope</td>
			<td style="width:152px;">Nikon&#160;</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">NIS Elements Advanced Research software</td>
			<td style="width:152px;">Nikon&#160;</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;">Imaris:Bitplane software version 7.6</td>
			<td style="width:152px;">Bitplane</td>
			<td style="width:127px;"></td>
			<td>&#34;coloc&#34; and &#34;supass&#34; modules are used.&#160;Alternatively, the open-source freeware&#160;ImageJ can be used for colocalization&#160;analysis of confocal z-stacks datasets.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td style="width:152px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;"></td>
			<td style="width:152px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:384px;"></td>
			<td style="width:152px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative 3d in silico modeling (q3dism) of cerebral amyloid-beta phagocytosis in rodent models of alzheimer's disease

Neuroinfiammazione è ormai riconosciuto come un importante fattore eziologico nella malattia neurodegenerativa. fagociti mononucleari sono cellule immunitarie innate responsabili della fagocitosi e la bonifica di detriti e detriti. Queste cellule sono macrofagi CNS residenti note come microglia, e fagociti mononucleari infiltrati dalla periferia. La microscopia ottica è stata generalmente utilizzati per la visualizzazione fagocitosi in roditori o campioni di cervello umano. Tuttavia, i metodi qualitativi non hanno fornito la prova definitiva di fagocitosi in vivo. Qui, descriviamo 3D quantitativa nella modellazione in silico (q3DISM), un metodo affidabile che permette una reale quantificazione 3D di amiloide-β (Ap) fagocitosi da parte dei fagociti mononucleari in modelli di roditori malattia di Alzheimer (AD). Il metodo consiste nel visualizzare fluorescente Ap incapsulato all&#39;interno phagolysosomes nelle sezioni roditore cervello. Grandi serie di dati confocale z-dimensionale vengono poi ricostruite in 3D per la quantificazione di A &amp;# 946; spazialmente colocalized all&#39;interno del fagolisosoma. Dimostriamo l&#39;applicazione di successo di q3DISM di topo e ratto cervello, ma questa metodologia può essere esteso a qualsiasi evento di fagocitosi in qualsiasi tessuto.

Utilizzando la metodologia più stadi per q3DISM dettagliato sopra, siamo in grado di quantificare l&#39;assorbimento Ap in phagolysosomes monociti nel cervello di APP / PS1 topi (figura 1) e ratti TgF344-AD (Figura 2). Pertanto, la metodologia q3DISM ha permesso l&#39;analisi dei fagociti mononucleari in modelli di topo e ratto di AD. È interessante notare che il volume occupato da CD68 + phagolysosomes è significativamente aumentata in fag...

Watch this Scientific Journal Video about Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease at JoVE.com

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Abbiamo sviluppato una metodologia per il 3D quantitativa in silico di modellazione (q3DISM) di cerebrale amiloide-β (Ap) fagocitosi da parte dei fagociti mononucleari in modelli di roditori della malattia di Alzheimer. Questo metodo può essere generalizzato per la quantificazione di praticamente qualsiasi evento fagocitaria in vivo.

3D Quantitative<em&gt; In Silico</em&gt; Modeling (q3DISM) di cerebrale amiloide-beta fagocitosi in roditori modelli di malattia di Alzheimer

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Research

JoVE Journal

Medicine

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Examining the Characteristics of Episodic Memory using Event-related Potentials in Patients with Alzheimer's Disease

Esaminando le caratteristiche della memoria episodica con potenziali evento-correlati nei pazienti con malattia di Alzheimer

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic ...

Ricerca

Medicina

Quantitative Analysis of Chromatin Proteomes in Disease

Analisi quantitativa di proteomi cromatina nella malattia

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Modelli di interazione gene-ambiente per smascherare i meccanismi di suscettibilità nel morbo di Parkinson

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Corsa di modellazione in Mouse: Coagulazione permanente dell&#39;arteria cerebrale media distale

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Multi-elettrodi array registrazioni di umano Epileptic postoperatoria corticale del tessuto

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

L&#39;uso di ultra-alto campo MRI in piccolo roditore modelli di malattia del rene policistico per<em&gt; In Vivo</em&gt; Fenotipizzazione and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

Ex Vivo intestinale Sacs per valutare mucosa Permeabilità in modelli di malattia gastrointestinale

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension

Emodinamica Caratterizzazione di roditori modelli di ipertensione arteriosa polmonare

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle ...

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

Neurodegenerazione in un modello animale di cronica amiloide-beta Oligomer infusione viene neutralizzata da anticorpi trattamento infuso con pompe osmotiche

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It ...

Application of Granger Causality Analysis of the Directed Functional Connection in Alzheimer's Disease and Mild Cognitive Impairment

Applicazione dell'analisi di causalità di Granger del diretto collegamento funzionale nella malattia di Alzheimer e Mild Cognitive Impairment

Impaired functional connectivity in the Default Mode Network (DMN) may be involved in the progression of Alzheimer&#39;s Disease (AD). The Posterior ...