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

Verklaring van het onderzoek ethiek: Alle experimenten met dieren die hierin beschreven werden goedgekeurd door de University of Southern California Institutional Animal Care en gebruik Comite (IACUC) en uitgevoerd in strikte overeenstemming met de National Institutes of Health richtlijnen en aanbevelingen van de Vereniging voor evaluatie en accreditatie van Laboratory Animal Care International. 1. knaagdieren Brain Isolatie en Voorbereiding voor Immunokleuring DAG 1: <ol><li> Plaats d...

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Het protocol dat we beschrijven in dit verslag voor echte 3D-kwantificering van Ap fagocytose in vivo door mononucleaire fagocyten steunt op specifieke etikettering van cellulaire en subcellulaire compartimenten evenals Ap-deposito&#39;s. Specifiek gebruiken we Iba1 (geïoniseerd-calcium bindende adapter molecuul 1), een eiwit dat betrokken is bij membraan roffelende en fagocytose na celactivering 18, 19, cerebrale mononucleaire fagocyten vlekken. Terwijl Iba1 + celle...

Ziekte van Alzheimer (AD), de meest voorkomende leeftijd gerelateerde dementie 1, gekenmerkt door cerebrale amyloïde-β (Aß) accumulatie &quot;seniele&quot; β-amyloïde plaques, chronische lage zenuwontsteking, tauopathie, neuronaal verlies en cognitieve stoornis 2 . In AD patiënt hersenen, wordt neuroinflammatie gereserveerd door reactieve astrocyten en mononucleaire fagocyten (aangeduid als microglia, hoewel hun centrale vs. perifere herkomst onduidelijk blijft) omliggende Aß afzettingen 3. Aangezien het aangeboren immuunsysteem wachters van het CZS, zijn microglia centraal gepositioneerd hersenen Aß wissen. Echter microglia werving Ap-plaques gepaard met zeer weinig of geen Aß fagocytose 4,5. Een hypothese is dat microglia in eerste instantie neuroprotectieve door phagocytozing kleine vergaderingen van AB. Echter, uiteindelijk deze cellen neurotoxisch overweldigend Aß last en / of leeftijdsgerelateerde functionele decline, provoceert microglia in een disfunctionele pro-inflammatoire fenotype, wat bijdraagt aan neurotoxiciteit en cognitieve achteruitgang 6. Recente Genoombrede Association Studies (GWAS) hebben een cluster van AD risico-allelen die behoren tot de belangrijkste aangeboren immuunsysteem trajecten 7 dat fagocytose 8-11 moduleren geïdentificeerd. Bijgevolg, de immuunrespons op cerebrale amyloïde afzetting is uitgegroeid tot een belangrijk aandachtsgebied, zowel in termen van het begrip van AD etiologie en voor het ontwikkelen van nieuwe therapeutische benaderingen 12-14. Toch is er een belangrijke behoefte aan methoden Ap fagocytose in vivo te evalueren. Om dit onvervulde behoefte aan te pakken, hebben we kwantitatieve 3D in silico modellering (q3DISM) naar echte 3D-kwantificering van cerebrale Ap fagocytose door mononucleaire fagocyten in knaagdier modellen van Alzheimer-achtige ziekte in te schakelen ontwikkeld. alleen beperkt door de mate waarin zij recapituleren ziekte, dierlijke modellen hebbenbewezen van onschatbare waarde voor het begrijpen van AD pathoetiology en voor het evalueren van experimentele therapieën. Vanwege het feit dat mutaties in de preseniline (PS) en Amyloid Precursor Protein (APP) genen onafhankelijk veroorzaken autosomaal dominante AD, zijn deze mutant transgenen uitgebreid toegepast om transgene knaagdiermodellen genereren. Transgene APP / PS1 muizen gelijktijdig co-expressie &quot;Swedish&quot; mutant menselijk APP (APP swe) en Δ exon 9 mutant menselijk preseniline 1 (PS1ΔE9) aanwezig met versnelde cerebrale amyloïdose en neuroinflammatie 15,16. Verder hebben we bi-transgene ratten gecoïnjecteerd met APP swe en PS1ΔE9 constructies (lijn TgF344-AD, op een Fischer 344 achtergrond) gegenereerd. In tegenstelling tot transgene muismodellen van cerebrale amyloïdose, TgF344-AD ratten ontwikkelen van cerebrale amyloïde dat tauopathie, apoptotische verlies van neuronen, en gedragsmatige impairment 17 voorafgaat. In dit rapport beschrijven we een protocol voor immunostaining microglia, phagolysosomes en Ap-afzettingen in de hersenen secties van APP / PS1 muizen en TgF344-AD ratten, en de verwerving van grote z-dimensionale confocale beelden. We detail in silico generatie en analyse van de echte 3D-reconstructies van confocale datasets waardoor kwantificering van Ap-opname in microglia phagolysosomes. Meer in het algemeen, kan de methodologie die detail Here We worden gebruikt om vrijwel elke vorm van fagocytose in vivo te kwantificeren.

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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>
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			<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>
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			<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>
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			<td height="20" style="height:20px;width:384px;">Tweezers</td>
			<td style="width:152px;">VWR</td>
			<td>94024-408</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>
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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>
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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>
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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>
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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>
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			<td height="20" style="height:20px;width:384px;">Embedding and Infiltration Paraffin</td>
			<td style="width:152px;">VWR</td>
			<td>15147-839</td>
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			<td height="20" style="height:20px;width:384px;">Microtome Leica RM2125</td>
			<td style="width:152px;">Leica Biosystems</td>
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		<tr height="20">
			<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>
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			<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>
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		<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>
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			<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>
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		<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>
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			<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>
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			<td height="20" style="height:20px;width:384px;">Triton X-100</td>
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			<td style="width:127px;">97062-208</td>
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			<td style="width:152px;">VWR</td>
			<td style="width:127px;">48393081</td>
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			<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>
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			<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>
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		<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>
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		<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>
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		<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

Neuro-inflammatie wordt nu erkend als een belangrijke etiologische factor in neurodegeneratieve ziekte. Mononucleaire fagocyten zijn aangeboren immuunsysteem cellen die verantwoordelijk zijn voor de fagocytose en het ruimen van puin en afval. Deze cellen zijn onder andere CNS-resident macrofagen bekend als microglia en mononucleaire fagocyten infiltreren uit de periferie. Lichtmicroscopie over het algemeen gebruikt om fagocytose visualiseren knaagdieren of menselijke hersenen specimens. Echter kwalitatieve methoden geen definitief bewijs van in vivo fagocytose. Hier beschrijven we kwantitatieve 3D in silico modellering (q3DISM), een robuuste methode waardoor echte 3D-kwantificering van amyloid-β (Aß) fagocytose door mononucleaire fagocyten in knaagdieren de ziekte van Alzheimer (AD) modellen. De methode houdt fluorescerend visualiseren Ap ingekapseld in phagolysosomes inproefdierhersenen secties. Grote z-dimensionale confocale datasets worden vervolgens 3D gereconstrueerd voor kwantificering van A &amp;# 946; ruimtelijk colocalized binnen de fagolysosoom. We tonen de succesvolle toepassing van q3DISM muizen- en ratten hersenen, maar deze methode kan worden uitgebreid tot vrijwel elke fagocytische gebeurtenis in elk weefsel.

Met behulp van de multi-stage methodologie voor q3DISM hierboven beschreven, zijn we in staat om Ap opname te kwantificeren in monocyten phagolysosomes in de hersenen van APP / PS1 muizen (figuur 1) en TgF344-AD ratten (figuur 2). Daarom is de methode q3DISM analyse van mononucleaire fagocyten in muis en ratmodellen van AD ingeschakeld. Interessant is dat het volume ingenomen door CD68 + phagolysosomes aanzienlijk toegenomen in Iba1 +</su...

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

We ontwikkelden een methode voor kwantitatieve 3D in silico modellering (q3DISM) van cerebrale amyloid-β (Aß) fagocytose door mononucleaire fagocyten in diermodellen van de ziekte van Alzheimer. Deze methode kan worden gegeneraliseerd voor de kwantificering van vrijwel elke fagocytische gebeurtenis in vivo.

kwantitatieve 3D<em&gt; In Silico</em&gt; Modeling (q3DISM) van cerebrale amyloid-beta Phagocytosis in diermodellen van de ziekte van 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

Het onderzoeken van de Kenmerken van episodisch geheugen met behulp van Event-related potentials bij patiënten met de ziekte van Alzheimer

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

Quantitative Analysis of Chromatin Proteomes in Disease

Kwantitatieve analyse van chromatine proteomen in de ziekte van

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

Gen-omgeving Interactiemodellen om gevoeligheidsmechanismen bij de ziekte van Parkinson te ontmaskeren

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

Modellering Stroke in Muizen: Permanente Coagulatie van de distale midden cerebrale slagader

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-electrode array Opnamen van Human Epileptische Postoperatieve Cortical Tissue

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

Het gebruik van ultra-hoge gebied MRI in kleine knaagdieren Modellen van Polycystic voor<em&gt; In Vivo</em&gt; Fenotypering 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 mucosale permeabiliteit beoordelen Modellen van gastro-intestinale aandoeningen

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

Hemodynamische karakterisatie van diermodellen van pulmonale arteriële hypertensie

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

Neurodegeneratie in een diermodel van chronische Amyloid-beta oligomeer infusie wordt tegengewerkt door antilichaambehandeling doordrongen osmotische pompen

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

Toepassing van Granger causaliteit analyse van de gestuurde functionele verbinding in de ziekte van Alzheimer en Mild cognitief stoornis

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