In vivo real-time dynamics of ATP and ROS production in axonal mitochondria show decoupling in mouse models of peripheral neuropathies

Background: Reactive oxygen species (ROS) play a central role in cigarette smoking-induced atherogenesis. The present study aims to assess the smoking-induced acute oxidative stress within vasculatures, and evaluates whether the resveratrol, a natural polyphenol antioxidant, can counteract this ROS production, using a customized, high resolution intravital optical imaging in real-time. Methods and Results: 20-week-old male C57BL/6 mice were divided into four groups according to the preceding administration of resveratrol (R) (25mg/kg via gavage, for 7 days) and exposure to cigarette smoke (CS). To in vivo assess acute oxidative stress in blood vessels, dihydroethidium, which forms a red fluorescence (ethidium, excitation/emission: 520nm/610nm) upon reaction with ROS, was injected intraperitoneally. During CS exposure, temporal changes of fluorescence signals from the mouse cremaster muscle including vasculatures were assessed by intravital optical imaging for 15 minutes. Fluorescence signals were much more pronounced in CS exposed mice than controls (p<0.001). Resveratrol p.o. significantly reduced the CS-induced ROS signals compared to the non-treated group (fluorescence signal to noise ratio, SNR, 2.51±0.09 vs. 12.52±2.116, p=0.0002) (Figure A). Without CS exposure, fluorescence signals in targeted vasculatures were very low showing no difference between groups (SNR, 1.65±0.19 vs. 1.53±0.07, p=0.80) (Figure A). Lipid peroxidation was increased in CS group and significantly attenuated in resveratrol-treated mice (Figure B). Fluorescence microscopy and immunostainings corroborated the in vivo findings. Conclusions: The intravital optical imaging was able to in vivo estimate the dynamic changes of ROS production by CS exposure. Our data demonstrated that even a brief exposure to CS increased oxidative stress in vasculatures promptly, and the resveratrol exerts protective effects against the CS-induced acute oxidative stress.

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Real-time analysis of T cell receptors in naive cells in vitro and in vivo reveals flexibility in synapse and signaling dynamics

Journal of Experimental Medicine ◽

10.1084/jem.20091201 ◽

2010 ◽

Vol 207 (12) ◽

pp. 2733-2749 ◽

Cited By ~ 66

Author(s):

Rachel S. Friedman ◽

Peter Beemiller ◽

Caitlin M. Sorensen ◽

Jordan Jacobelli ◽

Matthew F. Krummel

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

Real Time ◽

T Cell Activation ◽

Valuable Insight ◽

Time Dynamics ◽

Insight Into

The real-time dynamics of the T cell receptor (TCR) reflect antigen detection and T cell signaling, providing valuable insight into the evolving events of the immune response. Despite considerable advances in studying TCR dynamics in simplified systems in vitro, live imaging of subcellular signaling complexes expressed at physiological densities in intact tissues has been challenging. In this study, we generated a transgenic mouse with a TCR fused to green fluorescent protein to provide insight into the early signaling events of the immune response. To enable imaging of TCR dynamics in naive T cells in the lymph node, we enhanced signal detection of the fluorescent TCR fusion protein and used volumetric masking with a second fluorophore to mark the T cells expressing the fluorescent TCR. These in vivo analyses and parallel experiments in vitro show minimal and transient incorporation of TCRs into a stable central supramolecular activating cluster (cSMAC) structure but strong evidence for rapid, antigen-dependent TCR internalization that was not contingent on T cell motility arrest or cSMAC formation. Short-lived antigen-independent TCR clustering was also occasionally observed. These in vivo observations demonstrate that varied TCR trafficking and cell arrest dynamics occur during early T cell activation.

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Modeling Immune Checkpoint Inhibitor Efficacy in Syngeneic Mouse Tumors in an Ex Vivo Immuno-Oncology Dynamic Environment

International Journal of Molecular Sciences ◽

10.3390/ijms21186478 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6478

Author(s):

Daniel T. Doty ◽

Julia Schueler ◽

Vienna L. Mott ◽

Cassie M. Bryan ◽

Nathan F. Moore ◽

...

Keyword(s):

Real Time ◽

Mouse Models ◽

Immune Checkpoint ◽

Checkpoint Inhibitors ◽

Ex Vivo ◽

Confocal Imaging ◽

Checkpoint Blockade ◽

Syngeneic Mouse

The immune checkpoint blockade represents a revolution in cancer therapy, with the potential to increase survival for many patients for whom current treatments are not effective. However, response rates to current immune checkpoint inhibitors vary widely between patients and different types of cancer, and the mechanisms underlying these varied responses are poorly understood. Insights into the antitumor activities of checkpoint inhibitors are often obtained using syngeneic mouse models, which provide an in vivo preclinical basis for predicting efficacy in human clinical trials. Efforts to establish in vitro syngeneic mouse equivalents, which could increase throughput and permit real-time evaluation of lymphocyte infiltration and tumor killing, have been hampered by difficulties in recapitulating the tumor microenvironment in laboratory systems. Here, we describe a multiplex in vitro system that overcomes many of the deficiencies seen in current static histocultures, which we applied to the evaluation of checkpoint blockade in tumors derived from syngeneic mouse models. Our system enables both precision-controlled perfusion across biopsied tumor fragments and the introduction of checkpoint-inhibited tumor-infiltrating lymphocytes in a single experiment. Through real-time high-resolution confocal imaging and analytics, we demonstrated excellent correlations between in vivo syngeneic mouse and in vitro tumor biopsy responses to checkpoint inhibitors, suggesting the use of this platform for higher throughput evaluation of checkpoint efficacy as a tool for drug development.

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Real-time dynamics of prostaglandin F2α release from uterus and corpus luteum during spontaneous luteolysis in the cow

Reproduction ◽

10.1530/rep.1.00183 ◽

2004 ◽

Vol 128 (2) ◽

pp. 189-195 ◽

Cited By ~ 24

Author(s):

Koumei Shirasuna ◽

Hitomi Asaoka ◽

Tomas J Acosta ◽

Missaka P B Wijayagunawardane ◽

Masayuki Ohtani ◽

...

Keyword(s):

Corpus Luteum ◽

Real Time ◽

Prostaglandin F2α ◽

Paracrine Factor ◽

Time Dynamics ◽

Physiological Relevance ◽

A Site ◽

Local Release ◽

Venous Plasma

Prostaglandin (PG) F2α released from the uterus in a pulsatile fashion is essential to induce regression of the corpus luteum (CL) in the cow. In addition to the uterus, the CL has also been recognized as a site of PGF2α production. Therefore, this study aimed to determine the detailed dynamics of the releasing profile of CL-derived PGF2α together with uterus-derived PGF2α during spontaneous luteolysis in the cow. Non-lactating Holstein cows (n = 6) were surgically implanted with a microdialysis system (MDS) on day 15 (oestrus = day 0) of the oestrous cycle. Simultaneously, catheters were implanted to collect ovarian venous plasma ipsilateral to the CL as well as jugular venous plasma. The concentrations of PGF2α, 13,14-dihydro-15-keto-PGF2α (PGFM) and progesterone in the MDS and plasma samples were determined by enzyme immunoassays. The intra-luteal PGF2α secretion slightly increased after the onset of luteolysis (0 h) and drastically increased from 24 h, and was maintained at high levels towards the following oestrus. Furthermore, PGF2α was released from the CL into the ovarian vein in a pulsatile manner during spontaneous luteolysis. Also, the fact that intra-luteal secretion of PGF2α and PGFM showed a positive correlation indicates the existence of a local metabolic pathway for PGF2α in the CL. In conclusion, the present study clarified the real-time dynamics of uterus-derived PGF2α and CL-derived PGF2α during spontaneous luteolysis in the cow, and gives the first in vivo evidence that the CL releases PGF2α during spontaneous luteolysis in the cow. Although the physiological relevance of CL-derived PGF2α appears to be restricted to a local role as an autocrine/paracrine factor in the CL, overall results support the concept that the local release of PGF2α within the regressing CL amplifies the luteolytic action of PGF2α from the uterus.

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A kinase translocation reporter reveals real-time dynamics of ERK activity in Drosophila

10.1101/2022.01.07.475336 ◽

2022 ◽

Author(s):

Alice C Yuen ◽

Anadika R Prasad ◽

Vilaiwan M Fernandes ◽

Marc Amoyel

Keyword(s):

Real Time ◽

Growth Factor Receptor ◽

Time Lapse ◽

High Temporal Resolution ◽

Time Dynamics ◽

Extracellular Signal ◽

Genetic Perturbations ◽

Recent Developments ◽

Erk Activity

Extracellular Signal-Regulated Kinase (ERK) lies downstream of a core signalling cascade that controls all aspects of development and adult homeostasis. Recent developments have led to new tools to image and manipulate the pathway. However, visualising ERK activity in vivo with high temporal resolution remains a challenge in Drosophila. We adapted a kinase translocation reporter (KTR) for use in Drosophila, which shuttles out of the nucleus when phosphorylated by ERK. We show that ERK-KTR faithfully reports endogenous ERK signalling activity in developing and adult tissues, and that it responds to genetic perturbations upstream of ERK. Using ERK-KTR in time-lapse imaging, we made two novel observations: firstly, sustained hyperactivation of ERK by expression of dominant-active Epidermal Growth Factor Receptor raised the overall level but did not alter the kinetics of ERK activity; secondly, heterogeneity in ERK activity in retinal basal glia correlated with the direction of migration of individual cells. Our results show that KTR technology can be applied in Drosophila to monitor ERK activity in real-time and suggest that this modular tool can be further adapted to study other kinases.

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