A semi-automated cell tracking protocol for quantitative analyses of neutrophil swarming to sterile and S. aureus contaminated bone implants in a mouse femur model.

Autor: Lekkala S; Department of Biomedical Engineering, University of Rochester, Rochester, New York, United States of America.; Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York, United States of America., Ren Y; Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York, United States of America.; Department of Orthopaedics and Rehabilitation, University of Rochester Medical Center, Rochester, New York, United States of America., Weeks J; Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York, United States of America., Lee K; Department of Biomedical Engineering, University of Rochester, Rochester, New York, United States of America.; Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York, United States of America., Tay AJH; Department of Biomedical Engineering, University of Rochester, Rochester, New York, United States of America.; Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York, United States of America., Liu B; Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York, United States of America., Xue T; Department of Biomedical Engineering, University of Rochester, Rochester, New York, United States of America.; Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York, United States of America., Rainbolt J; Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York, United States of America., Xie C; Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York, United States of America.; Department of Orthopaedics and Rehabilitation, University of Rochester Medical Center, Rochester, New York, United States of America., Schwarz EM; Department of Biomedical Engineering, University of Rochester, Rochester, New York, United States of America.; Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York, United States of America.; Department of Orthopaedics and Rehabilitation, University of Rochester Medical Center, Rochester, New York, United States of America., Yeh SA; Department of Biomedical Engineering, University of Rochester, Rochester, New York, United States of America.; Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York, United States of America.; Department of Orthopaedics and Rehabilitation, University of Rochester Medical Center, Rochester, New York, United States of America.; Department of Physiology/Pharmacology, University of Rochester Medical Center, Rochester, New York, United States of America.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2024 Jun 20; Vol. 19 (6), pp. e0296140. Date of Electronic Publication: 2024 Jun 20 (Print Publication: 2024).
DOI: 10.1371/journal.pone.0296140
Abstrakt: Implant-associated osteomyelitis remains a major orthopaedic problem. As neutrophil swarming to the surgical site is a critical host response to prevent infection, visualization and quantification of this dynamic behavior at the native microenvironment of infection will elucidate previously unrecognized mechanisms central to understanding the host response. We recently developed longitudinal intravital imaging of the bone marrow (LIMB) to visualize host cells and fluorescent S. aureus on a contaminated transfemoral implant in live mice, which allows for direct visualization of bacteria colonization of the implant and host cellular responses using two-photon laser scanning microscopy. To the end of rigorous and reproducible quantitative outcomes of neutrophil swarming kinetics in this model, we developed a protocol for robust segmentation, tracking, and quantifications of neutrophil dynamics adapted from Trainable Weka Segmentation and TrackMate, two readily available Fiji/ImageJ plugins. In this work, Catchup mice with tdTomato expressing neutrophils received a transfemoral pin with or without ECFP/EGFP-expressing USA300 methicillin-resistant Staphylococcus aureus (MRSA) to obtain 30-minute LIMB videos at 2-, 4-, and 6-hours post-implantation. The developed semi-automated neutrophil tracking protocol was executed independently by two users to quantify the distance, displacement, speed, velocity, and directionality of the target cells. The results revealed high inter-user reliability for all outcomes (ICC > 0.96; p > 0.05). Consistent with the established paradigm on increased neutrophil swarming during active infection, the results also demonstrated increased neutrophil speed and velocity at all measured time points, and increased displacement at later time points (6 hours) in infected versus uninfected mice (p < 0.05). Neutrophils and bacteria also exhibit directionality during migration in the infected mice. The semi-automated cell tracking protocol provides a streamlined approach to robustly identify and track individual cells across diverse experimental settings and eliminates inter-observer variability.
Competing Interests: The authors have declared that no competing interests exist.
(Copyright: © 2024 Lekkala et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
Databáze: MEDLINE
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