Intravital longitudinal wide-area imaging of dynamic bone marrow engraftment and multilineage differentiation through nuclear-cytoplasmic labeling

Ahn, Soyeon; Choe, Kibaek; Lee, Seunghun; Kim, Kangsan; Song, Eunjoo; Seo, Hyun-Ji; Kim, Injune; Kim, Pilhan

doi:10.1371/journal.pone.0187660

Cited by 16 publications

(19 citation statements)

References 48 publications

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“…Mice were anesthetized using intramuscular injections of a cocktail mixture of zoletil (30 mg/kg) and xylazine (10 mg/kg). The imaging analysis was conducted as previously described ( Ahn et al, 2017 ). All experiments were performed in duplicate using two independent OMV sample preparations and at least two mice for each experiments.…”

Section: Methodsmentioning

confidence: 99%

Delivery of Periodontopathogenic Extracellular Vesicles to Brain Monocytes and Microglial IL-6 Promotion by RNA Cargo

Choi

Lee

et al. 2020

Front. Mol. Biosci.

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Gram-negative bacterial extracellular vesicles (EVs), also known as outer membrane vesicles (OMVs), are secreted from bacterial cells and have attracted research attention due to their role in cell-to-cell communication. During OMV secretion, a variety of cargo such as extracellular RNA (exRNA) is loaded into the OMV. The involvement of exRNAs from a range of bacteria has been identified in several diseases, however, their mechanism of action has not been elucidated. We have recently demonstrated that OMVs secreted by the periodontopathogen Aggregatibacter actinomycetemcomitans can cross the blood–brain barrier (BBB) and that its exRNA cargo could promote the secretion of proinflammatory cytokines in the brain. However, it was unclear whether the brain immune cells could actually take up bacterial OMVs, which originate outside of the brain, in an appropriate immune response. In the present study, using monocyte-specific live CX3CR1-GFP mice, we visualized OMV-colocalized meningeal macrophages and microglial cells into which bacterial OMVs had been loaded and intravenously injected through tail veins. Our results suggested that meningeal macrophages uptake BBB-crossed OMVs earlier than do cortex microglia. BV2 cells (a murine microglia cell line) and exRNAs were also visualized after OMV treatment and their proinflammatory cytokine levels were observed. Interleukin (IL)-6 and NF-κB of BV2 cells were activated by A. actinomycetemcomitans exRNAs but not by OMV DNA cargo. Altogether, these findings indicate that OMVs can successfully deliver exRNAs into brain monocyte/microglial cells and cause neuroinflammation, implicating a novel pathogenic mechanism in neuroinflammatory diseases.

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Section: Methodsmentioning

confidence: 99%

Delivery of Periodontopathogenic Extracellular Vesicles to Brain Monocytes and Microglial IL-6 Promotion by RNA Cargo

Choi

Lee

et al. 2020

Front. Mol. Biosci.

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“…Raster-pattern laser-scanning was achieved by using a rotating polygonal mirror (MC-5; Lincoln Laser, Tecumseh, Canada) for x-axis scanning and a galvanomirror (6230H; Cambridge Technology, Bedford, MA, USA) for y-axis scanning as previously described. 11 12 13 16 17 18 4f lens system was implemented by using anti-reflection coated achromatic lenses (L1:50mm FL, 49356INK, L2:100mm FL, 49360INK, L3:75mm FL, 49358INK, L4:200mm FL, 49364INK; Edmund Optics, Barrington, NJ, USA). The scanning laser beam was delivered to the liver of anesthetized mouse through commercial high numerical aperture objective lens (CFI75ApoLWD25XW, NA 1.1; Nikon, Tokyo, Japan).…”

Section: Methodsmentioning

confidence: 99%

Intravital Two-photon Imaging of Dynamic Alteration of Hepatic Lipid Droplets in Fasted and Refed State

Moon

Kim

2021

J Lipid Atheroscler

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Objective The liver plays a central role in lipid metabolism. During fasting and feeding, the fatty acid trafficking between adipose tissue and liver induces accumulation and dissociation of dynamic hepatic lipid droplets (LDs). Herein, we established an intravital 2-photon imaging technique to longitudinally visualize the dynamic in vivo alteration of hepatic LD deposition during fasting and refeeding in the liver of live mouse. Methods Intravital 2-photon imaging of liver was performed to observe hepatic LD alteration induced by fasting for different periods of time, 12, 24, and 48 hours followed by refeeding. Hepatic LDs were fluorescently labelled in vivo by intravenous injection of Seoul-Flour 44 and visualized by custom-built intravital 2-photon microscope. Results Significant increases of the number and size of hepatic LDs were observed by intravital 2-photon imaging of the liver after 12 hours of fasting. The degree of hepatic LD accumulation continuously increased with fasting up to 48 hours. Remarkably, with refeeding for 24 hours, the hepatic LDs accumulated by fasting were fully dissociated and the LD occupancy in the liver was recovered to the normal state. Conclusion Utilizing intravital 2-photon microscope with in vivo systemic fluorescent labeling of LD in live mice, dynamic alterations of hepatic LDs such as accumulation and dissociation by fasting and refeeding were successfully visualized at a subcellular level in vivo . The established method enabling the in vivo visualization of LDs will be a useful tool to investigate the pathophysiology of various diseases associated with dysregulated lipid metabolism.

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“…Custom-built video-rate laser-scanning confocal and two-photon microscope system previously implemented (Ahn, Kong et al, 2019, Ahn, Choe et al, 2017, Choe, Hwang et al, 2013, Lee, Kong et al, 2020, Park, Choe et al, 2018, Park, Kim et al, 2019, Seo, Hwang et al, 2015) was used. Four continuous-wave laser modules with output wavelengths at 405nm (OBIS 405, Coherent), 488 nm (MLD488, Cobolt), 561 nm (Jive, Cobolt) and 640 nm (MLD640, Cobolt) were used as an excitation sources for confocal microscope.…”

Section: Methodsmentioning

confidence: 99%

Intravital imaging of cerebral microinfarct reveals an astrocyte reaction led to glial scar

Hong

et al. 2021

Preprint

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Cerebral microinfarct increases the risk of dementia. But how microscopic cerebrovascular disruption affects the brain tissue in cellular-level are mostly unknown. Herein, with a longitudinal intravital imaging, we serially visualized in vivo dynamic cellular-level changes in astrocyte, pericyte and neuron as well as microvascular integrity after the induction of cerebral microinfarction for 1 month in mice. At day 2-3, it revealed a localized edema with acute astrocyte loss, neuronal death, impaired pericyte-vessel coverage and extravascular leakage indicating blood-brain barrier (BBB) dysfunction. At day 5, edema disappeared with recovery of pericyte-vessel coverage and BBB integrity. But brain tissue continued to shrink with persisted loss of astrocyte and neuron in microinfarct until 30 days, resulting in a collagen-rich fibrous scar surrounding the microinfarct. Notably, reactive astrocytes appeared at the peri-infarct area early at day 2 and thereafter accumulated in the peri-infarct. Oral administration of a reversible monoamine oxidase B inhibitor significantly decreased the astrocyte reactivity and fibrous scar formation. Our result suggests that astrocyte reactivity may be a key target to alleviate the impact of microinfarction.

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Intravital longitudinal wide-area imaging of dynamic bone marrow engraftment and multilineage differentiation through nuclear-cytoplasmic labeling

Cited by 16 publications

References 48 publications

Delivery of Periodontopathogenic Extracellular Vesicles to Brain Monocytes and Microglial IL-6 Promotion by RNA Cargo

Delivery of Periodontopathogenic Extracellular Vesicles to Brain Monocytes and Microglial IL-6 Promotion by RNA Cargo

Intravital Two-photon Imaging of Dynamic Alteration of Hepatic Lipid Droplets in Fasted and Refed State

Intravital imaging of cerebral microinfarct reveals an astrocyte reaction led to glial scar

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