In vivo real-time imaging of the liver with confocal endomicroscopy permits visualization of the temporospatial patterns of hepatocyte apoptosis

Apoptosis is a dynamic process of programmed cell death and is involved in multiple diseases. However, its mechanisms and sequence of events are still incompletely understood, partly because of the inability to visualize single cells continuously in vivo. The aim of the present study was to monitor hepatocyte apoptosis with confocal endomicroscopy in living rodents. In 73 anaesthetized mice, apoptotic liver injury was induced by injection of the CD95-agonistic antibody Jo2. Individual hepatocytes were followed for up to 240 min with a handheld confocal probe (FIVE1; Optiscan) providing 0.7 μm resolution (1,000-fold magnification). Different fluorescence staining protocols were used for cellular staining, vascular and cellular barrier function imaging, and caspase activation visualization. The time course of apoptosis could be visualized in vivo while liver perfusion and tissue integrity were maintained. In contrast to most ex vivo studies, initial cell swelling was observed that coincided with early defects in barrier function of sinusoids and hepatocytes. Cytoplasmic vesicle formation, nuclear condensation, cellular disintegration, and macrophage infiltration were captured sequentially. Labeling of caspases allowed molecular imaging. Our study allowed for the first time to continuously follow distinct morphological, functional, and molecular features of apoptosis in a solid organ in vivo and at high resolution. Intravital confocal microscopy may be a valuable tool to study the effects of therapeutic intervention on apoptosis in animal models and humans.

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In Vivo Real Time Imaging of Hepatocyte Apoptosis With Confocal Endomicroscopy Identifies Early Cell Swelling and Barrier Defect

Gastroenterology ◽

10.1016/s0016-5085(11)63841-9 ◽

2011 ◽

Vol 140 (5) ◽

pp. S-926

Author(s):

Martin Goetz ◽

Jacqueline V. Ansems ◽

Marcus Schuchmann ◽

Peter R. Galle ◽

Ralf Kiesslich

Keyword(s):

Real Time ◽

Cell Swelling ◽

Hepatocyte Apoptosis ◽

Real Time Imaging ◽

Confocal Endomicroscopy ◽

Barrier Defect

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In VivoRectal Mucosal Barrier Function Imaging in a Large-Animal Model by Using Confocal Endomicroscopy: Implications for Injury Assessment and Use in HIV Prevention Studies

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00134-16 ◽

2016 ◽

Vol 60 (8) ◽

pp. 4600-4609 ◽

Cited By ~ 1

Author(s):

Gracie Vargas ◽

Kathleen Listiak Vincent ◽

Yong Zhu ◽

David Szafron ◽

Tyra Caitlin Brown ◽

...

Keyword(s):

Animal Model ◽

Topical Application ◽

Barrier Function ◽

Mucosal Barrier ◽

Large Animal Model ◽

Large Animal ◽

Confocal Endomicroscopy ◽

Increased Risk ◽

Mucosal Barrier Function

ABSTRACTInjury occurring on the surface of the rectal mucosal lining that causes defects in barrier function may result in increased risk for transmission of infection by HIV and other pathogens. Such injury could occur from microbicidal or other topical agents, mechanical trauma during consensual or nonconsensual intercourse, or inflammatory conditions. Tools for evaluation of rectal mucosal barrier function for assessing the mucosa under these conditions are lacking, particularly those that can providein vivostructural and functional barrier integrity assessment and are adaptable to longitudinal imaging. We investigated confocal endomicroscopy (CE) as a means forin vivoimaging of the rectal epithelial barrier in the ovine model following spatially confined injury to the surface at a controlled site using a topical application of the microbicide test agent benzalkonium chloride. Topical and intravenous (i.v.) fluorescent probes were used with CE to provide subcellular resolution imaging of the mucosal surface and assessment of barrier function loss. A 3-point CE grading system based on cellular structure integrity and leakage of dye through the mucosa showed significant differences in score between untreated (1.19 ± 0.53) and treated (2.55 ± 0.75) tissue (P< 0.0001). Histological grading confirmed findings of barrier compromise. The results indicate that CE is an effective means for detecting epithelial injury and barrier loss following localized trauma in a large-animal model. CE is promising for real-time rectal mucosal evaluation after injury or trauma or topical application of emerging biomedical prevention strategies designed to combat HIV.

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Faculty Opinions recommendation of Epithelial barrier function in vivo is sustained despite gaps in epithelial layers.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1027900.334863 ◽

2005 ◽

Author(s):

Hannah Carey

Keyword(s):

Barrier Function ◽

Epithelial Barrier ◽

Epithelial Barrier Function ◽

Epithelial Layers

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Faculty Opinions recommendation of Ena/VASP is required for endothelial barrier function in vivo.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1095030.550015 ◽

2007 ◽

Author(s):

Giulio Gabbiani

Keyword(s):

Barrier Function ◽

Endothelial Barrier ◽

Endothelial Barrier Function

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Faculty Opinions recommendation of Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718235138.793512049 ◽

2015 ◽

Author(s):

Sarah Teichmann ◽

Valentina Proserpio

Keyword(s):

Single Cells ◽

Live Tissue ◽

In Vivo Analysis

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ATP potently modulates anion channel-mediated excitatory amino acid release from cultured astrocytes

AJP Cell Physiology ◽

10.1152/ajpcell.00438.2001 ◽

2002 ◽

Vol 283 (2) ◽

pp. C569-C578 ◽

Cited By ~ 91

Author(s):

Alexander A. Mongin ◽

Harold K. Kimelberg

Keyword(s):

Amino Acid ◽

Excitatory Amino Acid ◽

Atp Release ◽

Anion Channel ◽

P2y Receptors ◽

Cell Swelling ◽

Channel Blockers ◽

Medium Osmolarity ◽

Subsequent Activation

Volume-dependent ATP release and subsequent activation of purinergic P2Y receptors have been implicated as an autocrine mechanism triggering activation of volume-regulated anion channels (VRACs) in hepatoma cells. In the brain ATP is released by both neurons and astrocytes and participates in intercellular communication. We explored whether ATP triggers or modulates the release of excitatory amino acid (EAAs) via VRACs in astrocytes in primary culture. Under basal conditions exogenous ATP (10 μM) activated a small EAA release in 70–80% of the cultures tested. In both moderately (5% reduction of medium osmolarity) and substantially (35% reduction of medium osmolarity) swollen astrocytes, exogenous ATP greatly potentiated EAA release. The effects of ATP were mimicked by P2Y agonists and eliminated by P2Y antagonists or the ATP scavenger apyrase. In contrast, the same pharmacological maneuvers did not inhibit volume-dependent EAA release in the absence of exogenous ATP, ruling out a requirement of autocrine ATP release for VRAC activation. The ATP effect in nonswollen and moderately swollen cells was eliminated by a 5–10% increase in medium osmolarity or by anion channel blockers but was insensitive to tetanus toxin pretreatment, further supporting VRAC involvement. Our data suggest that in astrocytes ATP does not trigger EAA release itself but acts synergistically with cell swelling. Moderate cell swelling and ATP may serve as two cooperative signals in bidirectional neuron-astrocyte communication in vivo.

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Mechanism of CK2.3, a Novel Mimetic Peptide of Bone Morphogenetic Protein Receptor Type IA, Mediated Osteogenesis

International Journal of Molecular Sciences ◽

10.3390/ijms20102500 ◽

2019 ◽

Vol 20 (10) ◽

pp. 2500 ◽

Cited By ~ 3

Author(s):

Vrathasha Vrathasha ◽

Hilary Weidner ◽

Anja Nohe

Keyword(s):

Signaling Pathways ◽

Treatment Options ◽

Time Frame ◽

Bmp Signaling ◽

C2c12 Cells ◽

Molecular Sequence ◽

Sequence Of Events ◽

Protein Receptor

Background: Osteoporosis is a degenerative skeletal disease with a limited number of treatment options. CK2.3, a novel peptide, may be a potential therapeutic. It induces osteogenesis and bone formation in vitro and in vivo by acting downstream of BMPRIA through releasing CK2 from the receptor. However, the detailed signaling pathways, the time frame of signaling, and genes activated remain largely unknown. Methods: Using a newly developed fluorescent CK2.3 analog, specific inhibitors for the BMP signaling pathways, Western blot, and RT-qPCR, we determined the mechanism of CK2.3 in C2C12 cells. We then confirmed the results in primary BMSCs. Results: Using these methods, we showed that CK2.3 stimulation activated OSX, ALP, and OCN. CK2.3 stimulation induced time dependent release of CK2β from BMPRIA and concurrently CK2.3 colocalized with CK2α. Furthermore, CK2.3 induced BMP signaling depends on ERK1/2 and Smad1/5/8 signaling pathways. Conclusion: CK2.3 is a novel peptide that drives osteogenesis, and we detailed the molecular sequence of events that are triggered from the stimulation of CK2.3 until the induction of mineralization. This knowledge can be applied in the development of future therapeutics for osteoporosis.

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