Name: quantitative 3d in silico modeling (q3dism) of cerebral amyloid-beta phagocytosis in rodent models of alzheimer's disease
Uploaded: 2016-12-26T13:00:00
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;

Statement of research ethics: All experiments involving animals detailed herein were approved by the University of Southern California Institutional Animal Care and Use Committee (IACUC) and performed in strict accordance with National Institutes of Health guidelines and recommendations from the Association for Assessment and Accreditation of Laboratory Animal Care International.
1. Rodent Brain Isolation and Preparation for Immunostaining
DAY 1:
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	<li>Place aged TgF344-AD rats (14-mo...

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	<li>Brookmeyer, R., 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=National+estimates+of+the+prevalence+of+Alzheimer's+disease+in+the+United+States.">National estimates of the prevalence of Alzheimer's disease in the United States.</a> Alzheimers Dement. 7 (1), 61-73 (2011).</li><li>Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease.">Alzheimer's disease.</a> Cold Spring Harb Perspect Biol. 3 (7), (2011).</li><li>Heneka, M. T., Golenbock, D. T., Latz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immunity+in+Alzheimer's+disease.">Innate immunity in Alzheimer's disease.</a> Nat Immunol. 16 (3), 229-236 (2015).</li><li>Mawuenyega, , 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=Decreased+clearance+of+CNS+beta-amyloid+in+Alzheimer's+disease.">Decreased clearance of CNS beta-amyloid in Alzheimer's disease.</a> Science. 330 (6012), 1774 (2010).</li><li>Hickman, S. E., Allison, E. K., El Khoury, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+dysfunction+and+defective+beta-amyloid+clearance+pathways+in+aging+Alzheimer's+disease+mice.">Microglial dysfunction and defective beta-amyloid clearance pathways in aging Alzheimer's disease mice.</a> J Neurosci. 28 (33), 8354-8360 (2008).</li><li>Johnston, H., Boutin, H., Allan, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+contribution+of+inflammation+in+models+of+Alzheimer's+disease.">Assessing the contribution of inflammation in models of Alzheimer's disease.</a> Biochem Soc Trans. 39 (4), 886-890 (2011).</li><li>Gjoneska, E., 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=Conserved+epigenomic+signals+in+mice+and+humans+reveal+immune+basis+of+Alzheimer's+disease.">Conserved epigenomic signals in mice and humans reveal immune basis of Alzheimer's disease.</a> Nature. 518 (7539), 365-369 (2015).</li><li>Reitz, C., Mayeux, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer+disease:+epidemiology,+diagnostic+criteria,+risk+factors+and+biomarkers.">Alzheimer disease: epidemiology, diagnostic criteria, risk factors and biomarkers.</a> Biochem Pharmacol. 88 (4), 640-651 (2014).</li><li>Hazrati, L. -. N., 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=Genetic+association+of+CR1+with+Alzheimer's+disease:+a+tentative+disease+mechanism.">Genetic association of CR1 with Alzheimer's disease: a tentative disease mechanism.</a> Neurobiol Aging. 33 (12), 2949 (2012).</li><li>Griciuc, A., 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=Alzheimer's+Disease+Risk+Gene+CD33+Inhibits+Microglial+Uptake+of+Amyloid+Beta.">Alzheimer's Disease Risk Gene CD33 Inhibits Microglial Uptake of Amyloid Beta.</a> Neuron. , 1-13 (2013).</li><li>Li, X., Long, J., He, T., Belshaw, R., Scott, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+genomic+approaches+identify+major+pathways+and+upstream+regulators+in+late+onset+Alzheimer's+disease.">Integrated genomic approaches identify major pathways and upstream regulators in late onset Alzheimer's disease.</a> Scientific reports. 5, 12393 (2015).</li><li>Weitz, T. M., Town, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+in+Alzheimers+Disease:+"Its+All+About+Context".">Microglia in Alzheimers Disease: "Its All About Context".</a> Int J Alzheimers Dis. , 314185 (2012).</li><li>Guillot-Sestier, M. -. V., Doty, K. R., Town, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+Immunity+Fights+Alzheimer's+Disease.">Innate Immunity Fights Alzheimer's Disease.</a> Trends Neurosci. 38 (11), 674-681 (2015).</li><li>Guillot-Sestier, M. -. V., Town, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immunity+in+Alzheimer's+disease:+a+complex+affair.">Innate immunity in Alzheimer's disease: a complex affair.</a> CNS Neurol Disord Drug Targets. 12 (5), 593-607 (2013).</li><li>Jankowsky, J. L., Slunt, H. H., Ratovitski, T., Jenkins, N. A., Copeland, N. G., Borchelt, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-expression+of+multiple+transgenes+in+mouse+CNS:+a+comparison+of+strategies.">Co-expression of multiple transgenes in mouse CNS: a comparison of strategies.</a> Biomol Eng. 17 (6), 157-165 (2001).</li><li>Guillot-Sestier, M. -. 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=Il10+deficiency+rebalances+innate+immunity+to+mitigate+Alzheimer-like+pathology.">Il10 deficiency rebalances innate immunity to mitigate Alzheimer-like pathology.</a> Neuron. 85 (3), 534-548 (2015).</li><li>Cohen, R. 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=A+transgenic+Alzheimer+rat+with+plaques,+tau+pathology,+behavioral+impairment,+oligomeric+a&#946;,+and+frank+neuronal+loss.">A transgenic Alzheimer rat with plaques, tau pathology, behavioral impairment, oligomeric a&#946;, and frank neuronal loss.</a> J Neurosci. 33 (15), 6245-6256 (2013).</li><li>Imai, Y., Ibata, I., Ito, D., Ohsawa, K., Kohsaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+gene+iba1+in+the+major+histocompatibility+complex+class+III+region+encoding+an+EF+hand+protein+expressed+in+a+monocytic+lineage.">A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage.</a> Biochem. Biophys. Res. Commun. 224 (3), 855-862 (1996).</li><li>Ohsawa, K., Imai, Y., Sasaki, Y., Kohsaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia/macrophage-specific+protein+Iba1+binds+to+fimbrin+and+enhances+its+actin-bundling+activity.">Microglia/macrophage-specific protein Iba1 binds to fimbrin and enhances its actin-bundling activity.</a> J Neurochem. 88 (4), 844-856 (2004).</li><li>Bandyopadhyay, U., Nagy, M., Fenton, W. A., Horwich, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+lipofuscin+in+motor+neurons+of+SOD1-linked+ALS+mice.">Absence of lipofuscin in motor neurons of SOD1-linked ALS mice.</a> Proc Natl Acad Sci U S A. 111 (30), 11055-11060 (2014).</li><li>Holness, C. L., Simmons, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+CD68,+a+human+macrophage+marker+related+to+lysosomal+glycoproteins.">Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins.</a> Blood. 81 (6), 1607-1613 (1993).</li><li>Connor, 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=Phosphorylation+of+the+translation+initiation+factor+eIF2alpha+increases+BACE1+levels+and+promotes+amyloidogenesis.">Phosphorylation of the translation initiation factor eIF2alpha increases BACE1 levels and promotes amyloidogenesis.</a> Neuron. 60 (6), 988-1009 (2008).</li><li>Cai, 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=Phospholipase+D1+corrects+impaired+betaAPP+trafficking+and+neurite+outgrowth+in+familial+Alzheimer's+disease-linked+presenilin-1+mutant+neurons.">Phospholipase D1 corrects impaired betaAPP trafficking and neurite outgrowth in familial Alzheimer's disease-linked presenilin-1 mutant neurons.</a> Proc Natl Acad Sci U S A. 103 (6), 1936-1940 (2006).</li><li>Marsh, S. E., 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+adaptive+immune+system+restrains+Alzheimer's+disease+pathogenesis+by+modulating+microglial+function.">The adaptive immune system restrains Alzheimer's disease pathogenesis by modulating microglial function.</a> Proc Natl Acad Sci U S A. 113 (9), 1316-1325 (2016).</li><li>Lefterov, I., 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=Apolipoprotein+A-I+deficiency+increases+cerebral+amyloid+angiopathy+and+cognitive+deficits+in+APP/PS1DeltaE9+mice.">Apolipoprotein A-I deficiency increases cerebral amyloid angiopathy and cognitive deficits in APP/PS1DeltaE9 mice.</a> J Biol. Chem. 285 (47), 36945-36957 (2010).</li><li>Blurton-Jones, 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=Neural+stem+cells+improve+cognition+via+BDNF+in+a+transgenic+model+of+Alzheimer+disease.">Neural stem cells improve cognition via BDNF in a transgenic model of Alzheimer disease.</a> Proc Natl Acad Sci U S A. 106 (32), 13594-13599 (2009).</li><li>Stalder, M., Deller, T., Staufenbiel, M., Jucker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-Reconstruction+of+microglia+and+amyloid+in+APP23+transgenic+mice:+no+evidence+of+intracellular+amyloid.">3D-Reconstruction of microglia and amyloid in APP23 transgenic mice: no evidence of intracellular amyloid.</a> Neurobiol Aging. 22 (3), 427-434 (2001).</li><li>Leinenga, G., G&#246;tz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+ultrasound+removes+amyloid-&#946;+and+restores+memory+in+an+Alzheimer's+disease+mouse+model.">Scanning ultrasound removes amyloid-&#946; and restores memory in an Alzheimer's disease mouse model.</a> Sci Transl Med. 7 (278), 33 (2015).</li><li>Liarski, V. 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=Cell+distance+mapping+identifies+functional+T+follicular+helper+cells+in+inflamed+human+renal+tissue.">Cell distance mapping identifies functional T follicular helper cells in inflamed human renal tissue.</a> Sci Transl Med. 6 (230), 46 (2014).</li><li>Nichols, L., Pike, V. W., Cai, L., Innis, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+in+vivo+quantitation+of+beta-amyloid:+an+exemplary+biomarker+for+Alzheimer's+disease.">Imaging and in vivo quantitation of beta-amyloid: an exemplary biomarker for Alzheimer's disease.</a> Biol Psychiatry. 59 (10), 940-947 (2006).</li><li>Skovronsky, D. M., Zhang, B., Kung, M. P., Kung, H. F., Trojanowski, J. Q., Lee, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+detection+of+amyloid+plaques+in+a+mouse+model+of+Alzheimer's+disease.">In vivo detection of amyloid plaques in a mouse model of Alzheimer's disease.</a> Proc Natl Acad Sci U S A. 97 (13), 7609-7614 (2000).</li><li>Lian, H., Litvinchuk, A., Chiang, A. C. -. A., Aithmitti, N., Jankowsky, J. L., Zheng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-Microglia+Cross+Talk+through+Complement+Activation+Modulates+Amyloid+Pathology+in+Mouse+Models+of+Alzheimer's+Disease.">Astrocyte-Microglia Cross Talk through Complement Activation Modulates Amyloid Pathology in Mouse Models of Alzheimer's Disease.</a> J Neurosci. 36 (2), 577-589 (2016).</li><li>Novotny, R., 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=Conversion+of+Synthetic+A&#946;+to+In+Vivo+Active+Seeds+and+Amyloid+Plaque+Formation+in+a+Hippocampal+Slice+Culture+Model.">Conversion of Synthetic A&#946; to In Vivo Active Seeds and Amyloid Plaque Formation in a Hippocampal Slice Culture Model.</a> J Neurosci. 36 (18), 5084-5093 (2016).</li><li>Tartaro, K., 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=Development+of+a+fluorescence-based+in+vivo+phagocytosis+assay+to+measure+mononuclear+phagocyte+system+function+in+the+rat.">Development of a fluorescence-based in vivo phagocytosis assay to measure mononuclear phagocyte system function in the rat.</a> J Immunotoxicol. 12 (3), 239-246 (2015).</li></ol>

The protocol that we describe in this report for true 3D quantitation of A&#946; phagocytosis in vivo by mononuclear phagocytes relies on specific labeling of cellular and subcellular compartments as well as A&#946; deposits. Specifically, we use Iba1 (Ionized-calcium Binding Adaptor molecule 1), a protein that is involved in membrane ruffling and phagocytosis upon cell activation18,19, to stain cerebral mononuclear phagocytes. While Iba1+ cells are generally regarded ...

Alzheimer&#39;s Disease (AD), the most common age-related dementia1, is characterized by cerebral amyloid-&#946; (A&#946;) accumulation as &#34;senile&#34; &#946;-amyloid plaques, chronic low-level neuroinflammation, tauopathy, neuronal loss, and cognitive disturbance2. In AD patient brains, neuroinflammation is earmarked by reactive astrocytes and mononuclear phagocytes (referred to as microglia, although their central vs. peripheral origin remains unclear) surrounding A&#946; deposits3. As the innate immune sentinels of the CNS, microglia are centrally positioned to clear brain A&#946;. However, microglial recruitment to A&#946; plaques is accompanied by very little, if any, A&#946; phagocytosis4,5. One hypothesis is that microglia are initially neuroprotective by phagocytozing small assemblies of A&#946;. However, eventually these cells become neurotoxic as overwhelming A&#946; burden and/or age-related functional decline, provokes microglia into a dysfunctional proinflammatory phenotype, contributing to neurotoxicity and cognitive decline6.
Recent Genome-wide Association Studies (GWAS) have identified a cluster of AD risk alleles belonging to core innate immune pathways7 that modulate phagocytosis8-11. Consequently, the immune response to cerebral amyloid deposition has become a major area of interest, both in terms of understanding AD etiology and for developing new therapeutic approaches12-14. Yet, there is a vital need for methodology to evaluate A&#946; phagocytosis in vivo. To address this unmet need, we have developed quantitative 3D in silico modeling (q3DISM) to enable true 3D quantitation of cerebral A&#946; phagocytosis by mononuclear phagocytes in rodent models of Alzheimer-like disease.
Limited only by the extent to which they recapitulate disease, animal models have proven invaluable for understanding AD pathoetiology and for evaluating experimental therapeutics. Owing to the fact that mutations in the Presenilin&#160;(PS) and Amyloid Precursor Protein (APP) genes independently cause autosomal dominant AD, these mutant transgenes have been extensively used to generate transgenic rodent models. Transgenic APP/PS1 mice simultaneously coexpressing &#34;Swedish&#34; mutant human APP (APPswe) and &#916; exon 9 mutant human presenilin 1 (PS1&#916;E9) present with accelerated cerebral amyloidosis and neuroinflammation15,16. Further, we have generated bi-transgenic rats coinjected with APPswe and PS1&#916;E9 constructs (line TgF344-AD, on a Fischer 344 background). Unlike transgenic mouse models of cerebral amyloidosis, TgF344-AD rats develop cerebral amyloid that precedes tauopathy, apoptotic loss of neurons, and behavioral impairment17.
In this report, we describe a protocol for immunostaining microglia, phagolysosomes and A&#946; deposits in brain sections from APP/PS1 mice and TgF344-AD rats, and acquisition of large z-dimensional confocal images. We detail in silico generation and analysis of true 3D reconstructions from confocal datasets allowing quantitation of A&#946; uptake into microglial phagolysosomes. More broadly, the methodology that we detail here can be used to quantify virtually any form of phagocytosis 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>
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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>
			<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>
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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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			<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>
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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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			<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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			<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

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Using the multi-stage methodology for q3DISM detailed above, we are able to quantify A&#946; uptake into monocyte phagolysosomes in the brains of APP/PS1 mice (Figure 1) and TgF344-AD rats (Figure 2). Therefore, the q3DISM methodology has enabled analysis of mononuclear phagocytes in mouse and rat models of AD. Interestingly, the volume occupied by CD68+ phagolysosomes is significantly increased in Iba1+ mononuclear phagocytes associ...

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 developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

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

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

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Medicine

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