Abstract 9389: Thrombus Formation Processes are Dependent on Endothelial Injuries: Examined by in vivo Two-photon Molecular Imaging

Aim: The cellular mechanisms of thrombotic diseases associated with cardiovascular events remains unclear, largely because of an inability to visualize thrombus formation. Thus, we developed in vivo imaging technique based on single- and multi-photon microscopy and laser injuries technique to revealed the multicellular processes during thrombus development. Methods: We visualized the cell dynamics including single platelet behavior, and assessed dynamic cellular interplay in two thrombosis models using two photon microscopy to CAG-eGFP mice (Figure a, b). Results: First, we visualized that rapidly developing thrombi composed of discoid platelets without EC disruption was triggered by ROS induced by laser irradiation (Figure c). In this model, thrombus consisted by discoid platelet aggregations without leukocyte recruitment and not affected by Gr-1 antibody. The second model is, thrombus with EC disruption. High power laser induced EC erosion and extravasations of circulating leukocytes with thrombus development. Inflammatory cytokine, adhesion molecules dynamically control these two processes, and Gr-1 antibody significantly suppressed these steps. (Figure d) Leukocyte was immediately recruited into the subendothelial layers with bleeding and hemostatic reactions, and TLR4 signaling contributed to these steps. Pretreatmet of LPS or ischemic procedures markedly enhanced these steps. Thrombus included calcium activated cores and deformed platelets. Immigrated leukocyte also showed the increase of intracellular calcium. Summary: These results indicated that endothelial function, especially inflammatory status, determined the thrombotic reaction. Leukocyte also contributed with TLR4 signaling. In sum, using our imaging system can be a powerful tool to analyze thrombus formation and evaluate the therapeutic strategies.

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Thrombus Development Processes Are Dependent On Endothelial Injuries: Examined By In Vivo Molecular Imaging

Blood ◽

10.1182/blood.v122.21.1070.1070 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1070-1070

Author(s):

Satoshi Nishimura ◽

Koji Eto ◽

Ryozo Nagai

Keyword(s):

Adhesion Molecules ◽

Imaging System ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Late Phase ◽

Power Laser ◽

Cellular Mechanisms ◽

Cell Dynamics ◽

Moderate Power

Abstract The thrombotic cellular mechanisms associated with cardiovascular events remains unclear, largely because of an inability to visualize thrombus formation. In addition, the contribution of endothelial cell (EC) injuries to thrombus formation processes are unclear, and we developed in vivo imaging technique based on single- and multi-photon microscopy to revealed the multicellular processes during thrombus development (Figure a,b). We visualized the cell dynamics including single platelet behavior, and assessed dynamic cellular interplay in two thrombosis models. First, we visualized that rapidly developing thrombi composed of discoid platelets without EC disruption was triggered by ROS photochemically induced by moderate power laser irradiation (Figure c). In this model, thrombus consisted by discoid platelet aggregations without leukocyte recruitment. The second model is, thrombus with EC disruption. High power laser induced EC erosion and extravasations of circulating leukocytes with thrombus development. Inflammatory cytokine, adhesion molecules dynamically control these two processes. (Figure d)Figure.Figure. Using this technique, we elucidated that Lnk (adapter protein) regulates integrin signaling leading to stabilization of developing thrombus without EC disruption. Specifically, adhesion molecules dynamically control these processes. Thrombus formation was initiated by the binding of platelet GPIb-alpha to endothelial von Willebrand Factor in this model, and actin linker talin-dependent activation of alphaIIb-beta3 integrin in platelets was required for late phase thrombus stability. As for the thrombus formation with EC disruption, chemokine expressions in endothelium and leukocyte (especially neutrophils) recruitment played a significant role in these processes. TLR4 signaling also contributed to these steps. In sum, using our imaging system can be a powerful tool to analyze thrombus formation and evaluate the therapeutic strategies. Disclosures: No relevant conflicts of interest to declare.

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High-resolution two-photon transcranial imaging of brain using direct wavefront sensing

10.1101/2020.09.12.294421 ◽

2020 ◽

Author(s):

Congping Chen ◽

Zhongya Qin ◽

Sicong He ◽

Shaojun Liu ◽

Shun-Fat Lau ◽

...

Keyword(s):

High Resolution ◽

Adaptive Optics ◽

Pyramidal Neurons ◽

Imaging System ◽

Superficial Layer ◽

Wavefront Sensing ◽

Two Photon Microscopy ◽

Two Photon ◽

Transcranial Imaging

AbstractImaging of the brain in its native state at high resolution poses major challenges to visualization techniques. Two-photon microscopy integrated with the thinned-skull or optical clearing skull technique provides a minimally invasive tool for in vivo imaging of the cortex of mice without activating immune response and inducing brain injury. However, the imaging contrast and resolution are severely compromised by the optical heterogeneity of the skull, limiting the imaging depth to the superficial layer. Here, we develop adaptive optics two-photon microscopy for high-resolution transcranial imaging of layer 5 pyramidal neurons up to 700 μm below pia in living mice. In particular, an optimized configuration of imaging system and new wavefront sensing algorithm are proposed for accurate correction for the aberrations induced by the skull window and brain tissue. We investigated microglia-plaque interaction in living brain of Alzheimer’s disease and demonstrated high-precision laser dendrotomy and single-spine ablation.

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In vivo imaging of liver injury and regeneration by functional two-photon microscopy

Zeitschrift für Gastroenterologie ◽

10.1055/s-0036-1597342 ◽

2016 ◽

Vol 54 (12) ◽

pp. 1343-1404

Author(s):

A Ghallab ◽

R Reif ◽

R Hassan ◽

AS Seddek ◽

JG Hengstler

Keyword(s):

Liver Injury ◽

In Vivo Imaging ◽

Two Photon Microscopy ◽

Two Photon

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Cellular correlates of gray matter volume changes in magnetic resonance morphometry identified by two-photon microscopy

Scientific Reports ◽

10.1038/s41598-021-83491-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Livia Asan ◽

Claudia Falfán-Melgoza ◽

Carlo A. Beretta ◽

Markus Sack ◽

Lei Zheng ◽

...

Keyword(s):

Magnetic Resonance ◽

Cell Density ◽

Gray Matter ◽

Gray Matter Volume ◽

Cell Nuclei ◽

Cellular Mechanisms ◽

Tissue Volume ◽

Two Photon ◽

Matter Volume

AbstractMagnetic resonance imaging (MRI) of the brain combined with voxel-based morphometry (VBM) revealed changes in gray matter volume (GMV) in various disorders. However, the cellular basis of GMV changes has remained largely unclear. We correlated changes in GMV with cellular metrics by imaging mice with MRI and two-photon in vivo microscopy at three time points within 12 weeks, taking advantage of age-dependent changes in brain structure. Imaging fluorescent cell nuclei allowed inferences on (i) physical tissue volume as determined from reference spaces outlined by nuclei, (ii) cell density, (iii) the extent of cell clustering, and (iv) the volume of cell nuclei. Our data indicate that physical tissue volume alterations only account for 13.0% of the variance in GMV change. However, when including comprehensive measurements of nucleus volume and cell density, 35.6% of the GMV variance could be explained, highlighting the influence of distinct cellular mechanisms on VBM results.

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Sensory Transmission is Bi-directionally Modulated by Astrocytic Ca2+ in Barrel Cortex of Behaving Mice

10.21203/rs.3.rs-199750/v1 ◽

2021 ◽

Author(s):

Simeng Gu ◽

Wei Wang ◽

Kuan Zhang ◽

Rou Feng ◽

Naling Li ◽

...

Keyword(s):

Sensory Input ◽

Barrel Cortex ◽

Synaptic Function ◽

Metabolic Clearance ◽

Sleep State ◽

Two Photon Microscopy ◽

Two Photon ◽

Sensory Transmission

Abstract Different effects of astrocyte during sleep and awake have been extensively studied, especially for metabolic clearance by the glymphatic system, which works during sleep and stops working during waking states. However, how astrocytes contribute to modulation of sensory transmission during sleep and awake animals remain largely unknown. Recent advances in genetically encoded Ca2+ indicators have provided a wealth of information on astrocytic Ca2+, especially in their fine perisynaptic processes, where astrocytic Ca2+ most likely affects the synaptic function. Here we use two-photon microscopy to image astrocytic Ca2+ signaling in freely moving mice trained to run on a wheel in combination with in vivo whole-cell recordings to evaluate the role of astrocytic Ca2+ signaling in different behavior states. We found that there are two kinds of astrocytic Ca2+ signaling: a small long-lasting Ca2+ increase during sleep state and a sharp widespread but short-long-lasting Ca2+ spike when the animal was awake (fluorescence increases were 23.2 ± 14.4% for whisker stimulation at sleep state, compared with 73.3 ± 11.7% for at awake state, paired t-test, p < 0.01). The small Ca2+ transients decreased extracellular K+, hyperpolarized the neurons, and suppressed sensory transmission; while the large Ca2+ wave enhanced sensory input, contributing to reliable sensory transmission in aroused states. Locus coeruleus activation works as a switch between these two kinds of astrocytic Ca2+ elevation. Thus, we show that cortical astrocytes play an important role in processing of sensory input. These two types of events appear to have different pharmacological sources and may play a different role in facilitating the efficacy of sensory transmission.

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