研究目的
To quantify the effect of doses delivered by a He:Ne laser on individual macrophage kinetics, tissue oxidative stress, and wound closure using real-time in vivo imaging.
研究成果
A fluence of 3 J/cm2 was optimal for enhancing macrophage migration, reducing oxidative stress, and accelerating wound closure. The zebrafish model is effective for real-time quantitative analysis of photobiomodulation effects, suggesting potential for clinical applications in inflammatory conditions.
研究不足
The study focused on initial macrophage recruitment and may not capture long-term effects. The zebrafish model, while powerful, may have limitations in direct translation to mammalian systems. The range of fluences tested (3, 9, 18 J/cm2) might not cover all possible optimal parameters.
1:Experimental Design and Method Selection:
The study used a zebrafish model to investigate the effects of different laser fluences on macrophage behavior and oxidative stress in real-time. Methods included laser irradiation, time-lapse imaging, and wound closure assays.
2:Sample Selection and Data Sources:
Larval zebrafish Tg(mpeg-dendra2) were used, selected based on fluorescence, normal physiology, and absence of pre-existing wounds. Data were acquired through confocal microscopy and image analysis.
3:List of Experimental Equipment and Materials:
Equipment included a He:Ne laser (Zerona Medical Laser, Model EML-2245), confocal microscope (Zeiss LSM 510), and software (ZEN, MATLAB R2015a, ImageJ). Materials included zebrafish, dihydroethidium (DHE), and various chemicals.
4:Experimental Procedures and Operational Workflow:
Zebrafish were injured, treated with laser at specified fluences, and imaged at intervals. Oxidative stress was measured using DHE, and wound closure was assessed at 1 and 24 hours post-injury.
5:Data Analysis Methods:
Data were analyzed using Zirmi software in MATLAB for cell tracking, and statistical analyses were performed with GraphPad Prism and Microsoft Excel, including t-tests and linear regression.
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confocal microscope
LSM 510
Zeiss
Used for time-lapse imaging of zebrafish to capture macrophage kinetics and oxidative stress.
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software
ZEN
Zeiss
Used to acquire and control imaging parameters on the confocal microscope.
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He:Ne laser
EML-2245
Zerona Medical Laser, Erchonia Corporation
Used for photobiomodulation therapy, emitting 635 nm continuous wave at 5 mW output power to treat zebrafish wounds.
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software
MATLAB R2015a
MathWorks
Used with Zirmi software for analysis of time-lapse images and cell tracking.
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software
ImageJ
NIH
Used for measuring wound perimeters and analyzing wound closure.
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software
GraphPad Prism
GraphPad Software
Used for statistical analysis of experimental data.
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software
Microsoft Excel
Microsoft
Used for statistical analysis and data management.
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dihydroethidium
Sigma
Used as a probe for detecting oxidative stress in zebrafish tissues.
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Tricaine
Sigma
Used as an anesthetic for zebrafish during procedures.
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