Dataset Information

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

ABSTRACT: 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-? (A?) phagocytosis by mononuclear phagocytes in rodent Alzheimer's Disease (AD) models. The method involves fluorescently visualizing A? encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A? 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.

SUBMITTER: Guillot-Sestier MV

PROVIDER: S-EPMC5226459 | biostudies-other | 2016 Dec

REPOSITORIES: biostudies-other

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

Guillot-Sestier Marie-Victoire MV Weitz Tara M TM Town Terrence T

Journal of visualized experiments : JoVE 20161226 118

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 evid ...[more]

PMID: 28060279

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Project description:Traditionally, analysis of neuropathological markers in neurodegenerative diseases has relied on visual assessments of stained sections. Resulting semiquantitative scores often vary between individual raters and research centers, limiting statistical approaches. To overcome these issues, we have developed six deep learning-based models, that identify some of the most characteristic markers of Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). The deep learning-based models are trained to differentially detect parenchymal amyloid β (Aβ)-plaques, vascular Aβ-deposition, iron and calcium deposition, reactive astrocytes, microglia, as well as fibrin extravasation. The models were trained on digitized histopathological slides from brains of patients with AD and CAA, using a workflow that allows neuropathology experts to train convolutional neural networks (CNNs) on a cloud-based graphical interface. Validation of all models indicated a very good to excellent performance compared to three independent expert human raters. Furthermore, the Aβ and iron models were consistent with previously acquired semiquantitative scores in the same dataset and allowed the use of more complex statistical approaches. For example, linear mixed effects models could be used to confirm the previously described relationship between leptomeningeal CAA severity and cortical iron accumulation. A similar approach enabled us to explore the association between neuroinflammation and disparate Aβ pathologies. The presented workflow is easy for researchers with pathological expertise to implement and is customizable for additional histopathological markers. The implementation of deep learning-assisted analyses of histopathological slides is likely to promote standardization of the assessment of neuropathological markers across research centers, which will allow specific pathophysiological questions in neurodegenerative disease to be addressed in a harmonized way and on a larger scale.

Project description:BackgroundReactive microglia are associated with ?-amyloid (A?) deposit and clearance in Alzhiemer's Disease (AD). Paradoxically, entocranial resident microglia fail to trigger an effective phagocytic response to clear A? deposits although they mainly exist in an "activated" state. Oligomeric A? (oA?), a recent target in the pathogenesis of AD, can induce more potent neurotoxicity when compared with fibrillar A? (fA?). However, the role of the different A? forms in microglial phagocytosis, induction of inflammation and oxidation, and subsequent regulation of phagocytic receptor system, remain unclear.ResultsWe demonstrated that A?(1-42) fibrils, not A?(1-42) oligomers, increased the microglial phagocytosis. Intriguingly, the pretreatment of microglia with oA?(1-42) not only attenuated fA?(1-42)-triggered classical phagocytic response to fluorescent microspheres but also significantly inhibited phagocytosis of fluorescent labeled fA?(1-42). Compared with the fA?(1-42) treatment, the oA?(1-42) treatment resulted in a rapid and transient increase in interleukin 1? (IL-1?) level and produced higher levels of tumor necrosis factor-? (TNF-?), nitric oxide (NO), prostaglandin E2 (PGE2) and intracellular superoxide anion (SOA). The further results demonstrated that microglial phagocytosis was negatively correlated with inflammatory mediators in this process and that the capacity of phagocytosis in fA?(1-42)-induced microglia was decreased by IL-1?, lippolysaccharide (LPS) and tert-butyl hydroperoxide (t-BHP). The decreased phagocytosis could be relieved by pyrrolidone dithiocarbamate (PDTC), a nuclear factor-?B (NF-?B) inhibitor, and N-acetyl-L-cysteine (NAC), a free radical scavenger. These results suggest that the oA?-impaired phagocytosis is mediated through inflammation and oxidative stress-mediated mechanism in microglial cells. Furthermore, oA?(1-42) stimulation reduced the mRNA expression of CD36, integrin ?1 (Itgb1), and Ig receptor Fc?RIII, and significantly increased that of formyl peptide receptor 2 (FPR2) and scavenger receptor class B1 (SRB1), compared with the basal level. Interestingly, the pre-stimulation with oA?(1-42) or the inflammatory and oxidative milieu (IL-1?, LPS or t-BHP) significantly downregulated the fA?(1-42)-induced mRNA over-expression of CD36, CD47 and Itgb1 receptors in microglial cells.ConclusionThese results imply that A? oligomers induce a potent inflammatory response and subsequently disturb microglial phagocytosis and clearance of A? fibrils, thereby contributing to an initial neurodegenerative characteristic of AD. Antiinflammatory and antioxidative therapies may indeed prove beneficial to delay the progression of AD.

Dataset Information

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

Publications

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

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