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;

Erklæringen om forskningsetikk: Alle forsøk med dyr som er beskrevet i dette dokumentet ble godkjent av University of Southern California Institutional Animal Care og bruk Committee (IACUC) og utført i henhold til National Institutes of Health retningslinjer og anbefalinger fra Association for Assessment and Accreditation of Laboratory Animal Care International. 1. gnager Brain Isolasjon og forberedelse til Farging DAG 1: <ol><li> Plasser alderen TgF344-AD rotter (14 måneder gammel...

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Protokollen som vi beskriver i denne rapporten for ekte 3D kvantifisering av Ap fagocytose in vivo av mononukleære fagocytter er avhengig av spesifikk merking av cellulære og subcellulære avdelinger samt Ap-innskudd. Spesielt bruker vi Iba1 (ionisert kalsium Binding Adaptor molekyl 1), et protein som er involvert i membranen rusket og fagocytose på celleaktivering 18, 19, å farge cerebrale mononukleære fagocytter. Mens Iba1 + celler er generelt ansett som hjerne...

Alzheimers sykdom (AD), den mest vanlige aldersrelatert demens 1, karakterisert ved cerebral amyloid-β (Ap) akkumulering som &quot;senile&quot; p-amyloid plakk, kronisk lav-nivå nevroinflammasjon, tauopati, nevronale tap, og kognitiv forstyrrelse 2 . I AD pasient hjerner, er nevroinflammasjon øremerket av reaktive astrocytter og mononukleære fagocytter (referert til som microglia, selv om deres sentrale vs. perifer opprinnelse er fortsatt uklart) rundt Ap-innskudd 3. Som det medfødte immun voktere av CNS, er microglia sentralt plassert for å tømme hjernen Ap. Imidlertid er microglial rekrutteringen til Ap-plakk ledsaget av meget lite, om noen, Ap fagocytose 4,5. En hypotese er at microglia er i utgangspunktet nervecellene ved phagocytozing små forsamlinger av Ap. Imidlertid, etter hvert disse cellene blir nevrotoksisk som overveldende Ap belastning og / eller aldersrelaterte funksjonelle decline, provoserer microglia inn i en dysfunksjonell proinflammatoriske fenotype, bidra til nevrotoksisitet og kognitiv svikt 6. Siste Genome-wide Association Studies (GWAS) har identifisert en klynge av AD risiko alleler som hører til kjerne medfødte immun veier 7 som modulerer fagocytose 8-11. Derfor har immunresponsen til cerebral amyloid deponering blitt et stort område av interesse, både i forhold til å forstå AD etiologi og for å utvikle nye terapeutiske tilnærminger 12-14. Likevel er det et stort behov for metoder for å vurdere Ap fagocytose in vivo. For å møte dette udekket behov, har vi utviklet kvantitative 3D i silico modellering (q3DISM) for å aktivere ekte 3D kvantifisering av cerebral Ap fagocytose av mononukleære fagocytter i gnagermodeller av Alzheimer-lignende sykdom. Bare begrenset av i hvilken grad de rekapitulere sykdom, dyremodeller harvist seg uvurderlig for å forstå AD pathoetiology og for å vurdere eksperimentelle behandlingsformer. På grunn av det faktum at mutasjoner i presenilin (PS) og Amyloid Precursor Protein (APP) gener uavhengig forårsake autosomal dominant AD, har disse mutante transgener blitt mye brukt for å generere transgene gnagermodeller. Transgene APP / PS1 mus samtidig coexpressing &quot;svenske&quot; mutant menneskelige APP (APP swe) og Δ ekson 9 mutant human presenilin 1 (PS1ΔE9) til stede med akselerert cerebral amyloidose og nevroinflammasjon 15,16. Videre har vi generert bi-transgene rotter coinjected med APP swe og PS1ΔE9 konstruksjoner (linje TgF344-AD, på en Fischer 344 bakgrunnen). I motsetning til transgene musemodeller av cerebral amyloidose, TgF344-AD rotter utvikle cerebral amyloid som står foran tauopati, apoptotisk tap av nerveceller, og adferdsvansker 17. I denne rapporten beskriver vi en protokoll for immunostaining microglia, phagolysosomes og Ap-innskudd i hjernen seksjoner fra APP / PS1 mus og TgF344-AD rotter og oppkjøp av store z-dimensjonale confocal bilder. Vi detalj i silico generasjon og analyse av ekte 3D rekonstruksjoner fra confocal datasett tillater kvantifisering av Ap opptak i mikrogliaceller phagolysosomes. I videre forstand, kan den metodikken som vi detalj her brukes for å kvantifisere nesten enhver form for fagocytose 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>
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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>
		</tr>
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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>
			<td></td>
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		<tr height="20">
			<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>
			<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>
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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>
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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>
			<td></td>
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			<td height="20" style="height:20px;width:384px;">Microtome Leica RM2125</td>
			<td style="width:152px;">Leica Biosystems</td>
			<td style="width:127px;"></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>
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		<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>
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			<td height="20" style="height:20px;width:384px;">Coverslips</td>
			<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>
			<td></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>
			<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

Nevroinflammasjon er nå anerkjent som en viktig årsaksfaktor i nevrodegenerativ sykdom. Mononukleære fagocytter er medfødte immunceller som er ansvarlige for fagocytose og rydding av rusk og detritus. Disse cellene har CNS-resident makrofager kjent som microglia og mononukleære fagocytter infiltrere fra periferien. Lysmikroskopi har generelt blitt brukt til å visualisere fagocytose i gnager eller menneskelige hjerne prøver. Imidlertid har kvalitative metoder ikke gitt definitive bevis for in vivo fagocytose. Her beskriver vi kvantitativ 3D i silico modellering (q3DISM), en robust metode som åpner for ekte 3D kvantifisering av amyloid-β (Ap) fagocytose av mononukleære fagocytter i gnager Alzheimers sykdom (AD) modeller. Fremgangsmåten involverer fluorescens-visualisering av Ap innkapslet i phagolysosomes i gnagerhjerneseksjoner. Store z-dimensjonale confocal datasett blir deretter 3D rekonstruert for kvantifisering av A &amp;# 946; romlig colocalized innenfor phagolysosome. Vi demonstrerer vellykket bruk av q3DISM til mus og rottehjerner, men denne metoden kan utvides til nesten alle phagocytic hendelse i alle vev.

Bruke flertrinns metodikk for q3DISM beskrevet ovenfor, er vi i stand til å kvantifisere Ap opptak i monocytter phagolysosomes i hjernen til APP / PS1 mus (figur 1) og TgF344-AD rotter (figur 2). Derfor har q3DISM metodikk aktivert analyse av mononukleære fagocytter i mus og rottemodeller av AD. Interessant er det volumet som opptas av CD68 + phagolysosomes signifikant økt i Iba1 + mononukleære fagocytter er forbundet med i forh...

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

Vi utviklet en metodikk for kvantitativ 3D i silico modellering (q3DISM) av cerebral amyloid-β (Ap) fagocytose av mononukleære fagocytter i gnagermodeller av Alzheimers sykdom. Denne fremgangsmåten kan generaliseres for kvantifisering av praktisk talt alle fagocytisk event in vivo.

kvantitativ 3D<em&gt; I Silico</em&gt; Modeling (q3DISM) av cerebral amyloid-beta Fagocytose i gnagermodeller av Alzheimers sykdom

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

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

Kvantitativ analyse av Chromatin Proteomes i sykdom

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-miljø interaksjonsmodeller for å avdekke følsomhetsmekanismer ved Parkinsons sykdom

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 i Mus: Permanent koagulering av Distal Middle Cerebral Artery

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-elektrode Array Recordings of Human Epileptiske Postoperativ Kortikal 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

Bruk av Ultra-high Feltet MR i smågnager modeller av Polycystisk nyresykdom for<em&gt; I Vivo</em&gt; Fenotyping 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 Tarm Sacs å vurdere slimhinner Permeabilitet i modeller av gastrointestinal sykdom

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

Hemodynamisk Karakterisering av gnagermodeller av pulmonal arteriell hypertensjon

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

Nevrodegenerasjon i en dyremodell av kronisk amyloid-beta oliqomer Infusion motvirkes av antistoffbehandlingen Infused med osmotiske pumper

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

Anvendelsen av Granger kausalitet analyse av regissert funksjonelle tilkoblingen i Alzheimers sykdom og Mild kognitiv svekkelse

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