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Faculty
Discipline/Specialty (Author 1)
Medical Education
Academic Level (Author 2)
Faculty
Discipline/Specialty (Author 2)
Neuroscience
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Biomedical ENGR/Technology/Computation
Abstract
Background: Reactive oxygen species (ROS) are highly reactive molecules essential for various biological processes such as cell signaling and homeostasis. However, excessive ROS production leads to oxidative stress, contributing to diseases like cancer, neurodegenerative disorders, and cardiovascular conditions. Traditional ROS detection methods lack sensitivity, specificity, and real-time spatial-temporal resolution. Recently, fluorescent probes have emerged as powerful tools for detecting and visualizing ROS in biological systems. These probes emit fluorescence upon reacting with specific ROS, allowing precise localization and quantification. Understanding ROS fluctuations during hypoxia and reoxygenation is crucial, especially in skeletal muscle cells. This study explores the dynamics of ROS in response to oxygen level changes using advanced fluorescent detection techniques.
Methods: This research focuses on advancements in fluorescent detection techniques for ROS, examining the application of various fluorescent probes. We study the interaction mechanisms, selectivity, sensitivity, and advantages of these probes over traditional methods. Specifically, isolated rodent skeletal muscle models were exposed to alternating hyperoxia and hypoxia. To assess hydrogen peroxide (H2O2) and superoxide formation, dihydrofluorescein (Hfluor) and hydroethidine (DHE) probes were used, respectively. Tissue fluorescence was monitored using reflected and transmitted light to provide quantitative ROS levels.
Results: Oxygenation Effects: During hypoxia, tissues loaded with Hfluor or DHE probes showed a significant increase in fluorescence, indicating elevated production of ROS-H2O2 and ROS-superoxide. Upon reoxygenation, the fluorescence signals promptly declined, suggesting that ROS levels are dynamically regulated by oxygen availability.
Oxidized Probe Fluorescence: To validate the specificity of the probes, experiments using the oxidized forms of Hfluor and DHE were conducted. These control experiments revealed similar fluorescence patterns to those observed during hypoxia, indicating potential procedural artifacts influencing the signals.
Antioxidant Trials: Introducing ROS-specific antioxidants to hypoxic tissues effectively reduced the hypoxia-induced fluorescence. This supports the hypothesis that the increased fluorescence during hypoxia is due to ROS production, as antioxidants neutralized the ROS and decreased fluorescence.
Conclusion: Fluorescent detection of ROS provides crucial insights into the complex roles of these molecules, offering new therapeutic strategies and improved disease management. This study highlights that reduced oxygen levels induce hydrogen peroxide formation in skeletal muscles, emphasizing the intricate dynamics of ROS during hypoxia and reoxygenation. The findings underscore the importance of considering methodological nuances when interpreting ROS signals, providing valuable insights into muscle responses to varying oxygen environments. Additionally, targeting ROS with antioxidants could mitigate oxidative stress-related damage during hypoxia, with potential therapeutic implications for diseases characterized by hypoxia and reoxygenation cycles.
This study underscores the potential of fluorescent probes in biological research and clinical diagnostics, particularly in understanding the dynamics of ROS in living organisms. By shining light on the invisible, these probes reveal the complex behavior of ROS, paving the way for new therapeutic approaches and better disease management.
Presentation Type
Poster
Recommended Citation
Zuo, Alex, "Shining Light on the Invisible: Fluorescent Detection of Reactive Oxygen Species" (2024). Research Colloquium. 97.
https://scholarworks.utrgv.edu/colloquium/2024/posters/97
Included in
Analytical, Diagnostic and Therapeutic Techniques and Equipment Commons, Biotechnology Commons
Shining Light on the Invisible: Fluorescent Detection of Reactive Oxygen Species
Background: Reactive oxygen species (ROS) are highly reactive molecules essential for various biological processes such as cell signaling and homeostasis. However, excessive ROS production leads to oxidative stress, contributing to diseases like cancer, neurodegenerative disorders, and cardiovascular conditions. Traditional ROS detection methods lack sensitivity, specificity, and real-time spatial-temporal resolution. Recently, fluorescent probes have emerged as powerful tools for detecting and visualizing ROS in biological systems. These probes emit fluorescence upon reacting with specific ROS, allowing precise localization and quantification. Understanding ROS fluctuations during hypoxia and reoxygenation is crucial, especially in skeletal muscle cells. This study explores the dynamics of ROS in response to oxygen level changes using advanced fluorescent detection techniques.
Methods: This research focuses on advancements in fluorescent detection techniques for ROS, examining the application of various fluorescent probes. We study the interaction mechanisms, selectivity, sensitivity, and advantages of these probes over traditional methods. Specifically, isolated rodent skeletal muscle models were exposed to alternating hyperoxia and hypoxia. To assess hydrogen peroxide (H2O2) and superoxide formation, dihydrofluorescein (Hfluor) and hydroethidine (DHE) probes were used, respectively. Tissue fluorescence was monitored using reflected and transmitted light to provide quantitative ROS levels.
Results: Oxygenation Effects: During hypoxia, tissues loaded with Hfluor or DHE probes showed a significant increase in fluorescence, indicating elevated production of ROS-H2O2 and ROS-superoxide. Upon reoxygenation, the fluorescence signals promptly declined, suggesting that ROS levels are dynamically regulated by oxygen availability.
Oxidized Probe Fluorescence: To validate the specificity of the probes, experiments using the oxidized forms of Hfluor and DHE were conducted. These control experiments revealed similar fluorescence patterns to those observed during hypoxia, indicating potential procedural artifacts influencing the signals.
Antioxidant Trials: Introducing ROS-specific antioxidants to hypoxic tissues effectively reduced the hypoxia-induced fluorescence. This supports the hypothesis that the increased fluorescence during hypoxia is due to ROS production, as antioxidants neutralized the ROS and decreased fluorescence.
Conclusion: Fluorescent detection of ROS provides crucial insights into the complex roles of these molecules, offering new therapeutic strategies and improved disease management. This study highlights that reduced oxygen levels induce hydrogen peroxide formation in skeletal muscles, emphasizing the intricate dynamics of ROS during hypoxia and reoxygenation. The findings underscore the importance of considering methodological nuances when interpreting ROS signals, providing valuable insights into muscle responses to varying oxygen environments. Additionally, targeting ROS with antioxidants could mitigate oxidative stress-related damage during hypoxia, with potential therapeutic implications for diseases characterized by hypoxia and reoxygenation cycles.
This study underscores the potential of fluorescent probes in biological research and clinical diagnostics, particularly in understanding the dynamics of ROS in living organisms. By shining light on the invisible, these probes reveal the complex behavior of ROS, paving the way for new therapeutic approaches and better disease management.