研究目的
To investigate the effects of systemic administration of Meth at low dose on retinal damage and understand the underlying mechanisms of pathology.
研究成果
Systemic administration of low-dose Meth causes retinal damage in CD1 mice through apoptosis, oxidative stress, and inflammation. The study provides a model for future analysis of potential therapies.
研究不足
The study does not clarify whether inflammation and oxidative stress cause apoptosis of retinal cells in parallel or in series. The potential for reversing damage by preventing either or both pathways is hypothesized but not proven.
1：Experimental Design and Method Selection:
CD1 mice were treated with 0.5 mg/kg or 1 mg/kg Meth by intra-peritoneal injection daily for 2 months, with saline-treated mice as negative control. Electroretinography (ERG) was used to test outer retinal function. Toluidine blue staining was used for retinal morphology and photoreceptor cell loss evaluation. Inflammatory factors were measured by ELISA. TUNEL assay was used for apoptosis detection. Real-time PCR and Western blot were applied for gene and protein expression analysis.
2：5 mg/kg or 1 mg/kg Meth by intra-peritoneal injection daily for 2 months, with saline-treated mice as negative control. Electroretinography (ERG) was used to test outer retinal function. Toluidine blue staining was used for retinal morphology and photoreceptor cell loss evaluation. Inflammatory factors were measured by ELISA. TUNEL assay was used for apoptosis detection. Real-time PCR and Western blot were applied for gene and protein expression analysis. Sample Selection and Data Sources:
2. Sample Selection and Data Sources: Six-week-old CD1 mice were used, divided into 3 groups (n=10 each) for saline, 0.5 mg/kg Meth, or 1 mg/kg Meth treatment.
3：5 mg/kg Meth, or 1 mg/kg Meth treatment. List of Experimental Equipment and Materials:
3. List of Experimental Equipment and Materials: Electrophysiology System (UTAS-E3000; LKC Technologies), In Situ Cell Death Detection Kit (Roche Applied Science), ELISA kits, ReadyPrep Protein Extraction Kit (Bio-Rad), mouse cytochrome C immunoassay kit (R&D Systems), fluorescent assay kit (R&D systems), Qiagen RNeasy reagents, SuperScript master mix (Biorad), SYBR green Supermix (Thermo Fisher), StepOne systems (Thermo Fisher).
4：Experimental Procedures and Operational Workflow:
Mice were treated with Meth or saline for 2 months. ERG was recorded after 1 and 2 months. Retinas were harvested for various assays including TUNEL staining, oxidative stress markers measurement, cytokine levels measurement, and gene and protein expression analysis.
5：Data Analysis Methods:
Data were analyzed with SAS 9.1 software. ERG data were analyzed by 2-way repeated ANOVA. Other comparisons were made by 1-way ANOVA. P < .05 was considered significant.
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Electrophysiology System
UTAS-E3000
LKC Technologies
Used to record ERG responses from the corneal surface with a series of stimulus intensities.
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In Situ Cell Death Detection Kit
Roche Applied Science
Used to detect apoptosis of photoreceptor cells.
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ReadyPrep Protein Extraction Kit
Bio-Rad
Used to grind retinas with ReadyPrep Mini Grinders in lysis buffer.
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mouse cytochrome C immunoassay kit
R&D Systems
Used to determine the concentration of cytochrome C.
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fluorescent assay kit
R&D systems
Used to determine the caspase activities in retinas.
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Qiagen RNeasy reagents
Qiagen
Used to extract RNA from retinas.
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SuperScript master mix
Biorad
Used to transcribe mRNA into complementary DNA.
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SYBR green Supermix
Thermo Fisher
Used to run qPCR on StepOne systems.
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StepOne systems
Thermo Fisher
Used to run qPCR.
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