Presentation Details
| In vivo volume electron microscopy analysis at the single cell level reveals regional increases in platelet shape dispersity across a mouse, jugular vein puncture wound thrombus Madhavi A.Ariyarathne1, Irina D.Pokrovskaya1, Oliver S.Zhao2, Richard D.Leapman2, Maria A.Aronova2, Brian Storrie1. 1University of Arkansas for Medical Sciences, Little Rock, AR, USA.2National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, USA |
Abstract
Background: Platelet ultrastructure analysis using high-resolution 3D imaging is crucial for understanding various stages of platelet activation. Traditional microscopy techniques like light microscopy and transmission electron microscopy (TEM) lack the ability to provide detailed 3D image sets. However, recent advances, particularly in serial block-face scanning electron microscopy (SBF-SEM), offer the potential to generate high-resolution 3D images within in vivo thrombi in experimental models such as mice. To date, quantitative and detailed 3D ultrastructural analyses of in vivo activation stages of platelets at the individual cell level have been lacking. Objective: The primary goal of this study was to conduct an in vivo comparative analysis of platelet activation stages in puncture wound thrombi using SBF-SEM. The study aimed to bridge the gap in understanding platelet activation stages by in situ 3D analysis in mouse models. The research sought to provide the first in vivo 3D census of platelet ultrastructure across different activation stages and different subregions within a wound thrombus. Method: The research utilized SBF-SEM to capture images of platelets within various subregions in a 5-minute puncture wound thrombus in a mouse. Four specific regions of interest (ROIs) within the thrombus were selected to represent different stages of thrombus formation, namely ROI_01 – loosely adherent platelets, ROI_02 – transition to tightly adherent platelets, ROI_03 – hole exposed platelet surface and ROI_04 – collagen associated platelets surface) (Figure 1). We used Amira software for processing SBF-SEM images from ROIs. Amira software was utilized to identify, render, and quantify condensed alpha granules, decondensed alpha granules, mitochondria, and identify different platelet structures (Figure 2). Results: Preliminary findings revealed that ~80% of platelets in ROI_01 maintained a discoid cell shape, while ROI_02 displayed significant variation in cell shape, notably with the presence of pseudopods (Figure 2). These pseudopods arise due to platelet activation-induced cellular reorganization and potentially aid in mediating platelet aggregation. Moreover, analysis of the surface area to volume ratio plot in ROI_01 revealed a clustered pattern, indicating uniform platelet shapes with minimal variation compared to the dispersed distribution observed in ROI_02 (Figure 2). Interestingly, both surface area and surface area to volume ratio were significantly higher (p <0.05) in ROI_02 compared to ROI_01 (Figure 2). Furthermore, our analysis highlighted distinctive characteristics of mouse condensed alpha granules and mitochondria emphasizing their variable, elongated, rod-like shapes (Figure 2). Conclusion: The observations initiate the detailed characterization and comparison of different platelet activation stages at the level of individual platelets under in vivo conditions using advanced imaging techniques. These insights will significantly contribute to understanding platelet behavior and function in physiological contexts, especially in response to activation triggers like puncture wounds in experimental mouse models. The behavior of pseudopods suggests a potential role in platelet aggregation, indicating avenues for further exploration into platelet activation mechanisms.
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No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, including photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author.