研究目的
To determine if optic nerve head astrocyte structural reactivity is axon-dependent in response to acutely elevated intraocular pressure.
研究成果
Early ONH astrocyte structural reactivity to elevated IOP involves both axon-dependent and independent responses, with implications for astrocytes as targets in neurodegenerative disorders. Future studies should explore longer time courses and additional models.
研究不足
The study has a relatively short time course of IOP elevation and follow-up, and the rodent ONH lacks a true lamina cribrosa, which may limit direct applicability to human glaucoma.
1：Experimental Design and Method Selection:
The study used a rat model combining retrobulbar optic nerve transection (ONT) to induce axon loss and controlled elevation of intraocular pressure (CEI) to induce astrocyte reactivity. Immunofluorescence labeling and confocal microscopy were employed to assess structural and molecular changes.
2：Sample Selection and Data Sources:
Thirty-nine 8- to 9-month-old male Brown Norway rats were used, randomized into groups for ONT, CEI, or combined procedures.
3：List of Experimental Equipment and Materials:
Equipment included confocal microscope (FV1000, Olympus), pressure sensor (Harvard Apparatus), tonometer (TonoLab, Icare Finland Oy), and various surgical tools and reagents such as ketamine, xylazine, antibodies (e.g., anti-bIII tubulin, anti-phosphorylated cortactin), and fluorescent tags.
4：Experimental Procedures and Operational Workflow:
Animals underwent ONT or CEI procedures, with euthanization at specified times. ONH sections were prepared, labeled, and imaged using confocal microscopy. Data were analyzed with FIJI software.
5：Data Analysis Methods:
Quantitative analysis of label intensities and structural orientation was performed using FIJI software, with statistical analysis via ANOVA and multiple comparisons tests in GraphPad Prism.
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confocal microscope
FV1000
Olympus
Used for imaging ONH sections with confocal microscopy to quantify label intensities and structural changes.
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software
FV10-ASW version 4.0
Olympus
Software for capturing confocal microscopy images.
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secondary antibody
Alexa 488-labeled goat anti-mouse/rabbit
ThermoFisher Scientific
Secondary antibody for immunofluorescence labeling.
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phalloidin
TRITC-labeled
Sigma-Aldrich Corp.
Fluorescent tag for labeling actin filaments.
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mounting media
Prolong Gold with DAPI
ThermoFisher Scientific
Used for mounting slides and staining nuclei.
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pressure sensor
Harvard Apparatus
Coupled with infusion line to confirm pressure during CEI experiments.
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tonometer
TonoLab
Icare Finland Oy
Used for independent measurements of intraocular pressure every 30 minutes during CEI.
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microscope
Olympus
Used for light microscopy to assess axon injury in retrobulbar nerves.
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software
FIJI
National Institute of Health
Image analysis software for quantifying orientation and fluorescence intensities.
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software
GraphPad Prism
GraphPad
Used for statistical analysis including ANOVA and multiple comparisons tests.
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antibody
Tuj1
Covance
Primary antibody against bIII tubulin for labeling axons.
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antibody
p-paxillin
Abcam
Primary antibody against phosphorylated paxillin for labeling focal adhesion mediators.
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antibody
p-cortactin
EMD Millipore
Primary antibody against phosphorylated cortactin for labeling actin mediators.
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antibody
complement C3 B-9
Santa Cruz Biotechnology
Primary antibody against complement C3.
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anesthesia
ketamine
JHP Pharmaceuticals
Used for systemic anesthesia in animals.
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anesthesia
xylazine
RXV
Used for systemic anesthesia in animals.
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anesthesia
acepromazine maleate
VET ONE
Used for systemic anesthesia in animals.
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topical anesthetic
proparacaine hydrochloride
Akorn
Applied to ocular surface for local anesthesia.
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analgesia
buprenorphine
Buprenex
Used for postsurgical analgesia.
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ointment
erythromycin
Perrigo
Applied to ocular surface post-surgery.
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