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Scanning laser-induced endothelial injury: a standardized and reproducible thrombosis model for intravital microscopy.

ABSTRACT: Vascular injury models are indispensable for studying thrombotic processes in vivo. Amongst the available methods for inducing thrombosis, laser-induced endothelial injury (LIEI) has several unique advantages. However, a lack of methodological standardization and expensive instrumentation remain significant problems decreasing reproducibility and impeding the adoption of LIEI in the wider scientific community. In this, study, we developed a standardized protocol for scanning laser-induced endothelial injury (scanning-LIEI) of murine mesenteric veins using the intrinsic 405 nm laser of a conventional laser scanning confocal microscope. We show that our model produces thrombi with prominent core-shell architectures and minimal radiation-related fluorescence artefacts. In comparison with previous methods, the scanning-LIEI model exhibits reduced experimental variability, enabling the demonstration of dose-response effects for anti-thrombotic drugs using small animal cohorts. Scanning-LIEI using the intrinsic 405 nm laser of a confocal laser scanning microscope represents a new method to induce standardized vascular injury with improved reproducibility of thrombus formation. The reduced need for instrument customisation and user experience means that this model could be more readily adopted in the research community.

SUBMITTER: Larsson P

PROVIDER: S-EPMC8913794 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Scanning laser-induced endothelial injury: a standardized and reproducible thrombosis model for intravital microscopy.

Larsson P P Tarlac V V Wang T-Y TY Bonnard T T Hagemeyer C E CE Hamilton J R JR Medcalf R L RL Cody S H SH Boknäs N N

Scientific reports 20220310 1

Vascular injury models are indispensable for studying thrombotic processes in vivo. Amongst the available methods for inducing thrombosis, laser-induced endothelial injury (LIEI) has several unique advantages. However, a lack of methodological standardization and expensive instrumentation remain significant problems decreasing reproducibility and impeding the adoption of LIEI in the wider scientific community. In this, study, we developed a standardized protocol for scanning laser-induced endoth ...[more]

PMID: 35273275

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Project description:BACKGROUND:Microscopic images are widely used in plant biology as an essential source of information on morphometric characteristics of the cells and the topological characteristics of cellular tissue pattern due to modern computer vision algorithms. High-resolution 3D confocal images allow extracting quantitative characteristics describing the cell structure of leaf epidermis. For some issues in the study of cereal leaves development, it is required to apply the staining techniques with fluorescent dyes and to scan rather large fragments consisting of several frames. We aimed to develop a tool for processing multi-frame multi-channel 3D images obtained from confocal laser scanning microscopy and taking into account the peculiarities of the cereal leaves staining. RESULTS:We elaborated an ImageJ-plugin LSM-W2 that allows extracting data on Leaf Surface Morphology from Laser Scanning Microscopy images. The plugin is a crucial link in a workflow for obtaining data on structural properties of leaf epidermis and morphological properties of epidermal cells. It allows converting large lsm-files (laser scanning microscopy) into segmented 2D/3D images or tables with data on cells and/or nuclei sizes. In the article, we also represent some case studies showing the plugin application for solving biological tasks. Namely the plugin is applied in the following cases: defining parameters of jigsaw-puzzle pattern for maize leaf epidermal cells, analysis of the pavement cells morphological parameters for the mature wheat leaf grown under control and water deficit conditions, initiation of cell longitudinal rows, and detection of guard mother cells emergence at the initial stages of the stomatal morphogenesis in the growth zone of a wheat leaf. CONCLUSION:The proposed plugin is efficient for high-throughput analysis of cellular architecture for cereal leaf epidermis. The workflow implies using inexpensive and rapid sample preparation and does not require the applying of transgenesis and reporter genetic structures expanding the range of species and varieties to study. Obtained characteristics of the cell structure and patterns further could act as a basis for the development and verification for spatial models of plant tissues formation mechanisms accounting for structural features of cereal leaves. AVAILABILITY:The implementation of this workflow is available as an ImageJ plugin distributed as a part of the Fiji project (FijiisjustImageJ: https://fiji.sc/ ). The plugin is freely available at https://imagej.net/LSM_Worker , https://github.com/JmanJ/LSM_Worker and http://pixie.bionet.nsc.ru/LSM_WORKER/ .

Project description:BackgroundThe cremaster arteriole laser-induced injury model is a powerful technique with which to investigate the molecular mechanisms that drive thrombus formation. This model is capable of direct visualization and quantification of accumulation of thrombus constituents, including both platelets and fibrin. However, a large degree of variability in platelet accumulation and fibrin formation is observed between thrombi. Strategies to understand this variability will enhance performance and standardization of the model. We determined whether ablation injury size contributes to variation in platelet accumulation and fibrin formation and, if so, whether incorporating ablation injury size into measurements reduces variation.MethodsThrombus formation was initiated by laser-induced injury of cremaster arterioles of mice (n=59 injuries). Ablation injuries within the vessel wall were consistently identified and quantified by measuring the length of vessel wall injury observed immediately following laser-induced disruption. Platelet accumulation and fibrin formation as detected by fluorescently-labeled antibodies were captured by digital intra-vital microscopy.ResultsLaser-induced disruption of the vessel wall resulted in ablation injuries of variable length (18-95 μm) enabling interrogation of the relationship between injury severity and thrombus dynamics. Strong positive correlations were observed between vessel injury length and both platelet and fibrin when the data are transformed as area under the curve (Spearman r = 0.80 and 0.76 respectively). Normalization of area under the curve measurements by injury length reduced intraclass coefficients of variation among thrombi and improved hypothesis testing when comparing different data sets.ConclusionsMeasurement of vessel wall injury length provides a reliable and robust marker of injury severity. Injury length can effectively normalize measurements of platelet accumulation and fibrin formation improving data interpretation and standardization.

Dataset Information

Scanning laser-induced endothelial injury: a standardized and reproducible thrombosis model for intravital microscopy.

Publications

Scanning laser-induced endothelial injury: a standardized and reproducible thrombosis model for intravital microscopy.

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