研究目的
Evaluating the preventative effect of photobiomodulation (PBM) preconditioning and its underlying mechanism on brain damage in a neonatal rat model of hypoxia-ischemia.
研究成果
PBM preconditioning effectively prevents cognitive impairment and brain damage in a neonatal HI model by maintaining mitochondrial dynamics, inhibiting mitochondrial fragmentation, suppressing cytochrome c release, and preventing neuronal apoptosis. This suggests potential for PBM as a non-invasive preventative treatment for hypoxic-ischemic encephalopathy, though further research is needed to optimize parameters and validate in humans.
研究不足
The study is limited to a neonatal rat model, which may not fully translate to human infants. The optimal PBM parameters (e.g., power density, dosage) for preconditioning in HI models are not fully explored, and further investigation is needed for human application. The mechanisms underlying PBM effects require more in-depth study.
1：Experimental Design and Method Selection:
The study involved two experiments. Experiment 1 determined the optimal time interval for PBM preconditioning by measuring ATP levels in normal rat brains at various times post-PBM. Experiment 2 tested the neuroprotective effects of PBM preconditioning in a HI model, including behavioral tests, histological assessments, and molecular analyses to investigate mechanisms related to mitochondrial function and apoptosis.
2：Sample Selection and Data Sources:
10-day-old neonatal Sprague-Dawley rats of both sexes were used. Animals were randomly divided into groups for control, HI, and PBM+HI conditions. Brain samples were collected at postnatal days 16 and 29 for analysis.
3：List of Experimental Equipment and Materials:
Equipment included a diode IR Laser System (808 M100, Dragon Lasers) for PBM, a microplate luminometer (PE Applied Biosystems) for ATP measurement, a Leica Rm2155 microtome for sectioning, confocal laser microscope (LSM510 Meta, Carl Zeiss), and various kits for staining and assays (e.g., NeuroTrace TM fluorescent Nissl stain, Click-iT Plus TUNEL assay kit). Materials included antibodies (e.g., anti-MAP2 from Santa Cruz), reagents (e.g., paraformaldehyde, sucrose), and software (e.g., Image J, ANY-maze video tracking software).
4：Experimental Procedures and Operational Workflow:
For HI model, right common carotid artery was ligated, followed by hypoxia exposure (6% oxygen for 2 hours). PBM was applied 6 hours before HI using specified laser parameters. Behavioral tests (Novel Object Recognition, Objection Location test, Barnes Maze Task) were conducted from P26 to P
5：Brain collection involved perfusion, fixation, sectioning, and homogenization for protein analysis. Staining and assays (e.g., Nissl staining, TUNEL staining, immunofluorescence, Western blotting) were performed according to standard protocols. Data Analysis Methods:
Data were analyzed using Image J for image quantification, SigmaStat software for statistical analysis (one-way ANOVA followed by Student-Newman Keul's test), and ANY-maze software for behavioral tracking. Results are expressed as means ± SD with significance at P < 0.05.
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Leica microtome
Rm2155
Leica
Used for cutting frozen brain sections.
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Confocal laser microscope
LSM510 Meta
Carl Zeiss
Used for capturing fluorescence images in immunofluorescence staining.
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Diode IR Laser System
808 M100
Dragon Lasers
Used for delivering photobiomodulation (PBM) treatment to the scalp of neonatal rats.
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Fiber optic cable and multi-mode coupler
SMA connector; Imeter 400um multi model
Dragon Lasers
Used to apply the laser spot to the scalp during PBM treatment.
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Microplate luminometer
PE Applied Biosystems
Used to measure light output for ATP quantification.
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AxioVision microscope system
Carl Zeiss
Used to acquire images of brain sections for infarct volume assessment.
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Synergy HT Microplate reader
BioTek Instruments Inc
Used to measure fluorescence in caspase activity assays.
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ANY-maze video tracking software
Stoelting
Used to record and analyze animal behavior in behavioral tests.
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SigmaStat software
Systat Software
Used for statistical analysis of data.
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Image J software
NIH
Used for image quantification and analysis in various assays.
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