研究目的
To test the hypothesis that mitochondrial respiration contributes to local changes in hydration involved in phototransduction-driven expansion of outer retina, as measured by structural responses on optical coherence tomography (OCT) and diffusion magnetic resonance imaging (MRI).
研究成果
The results support the hypothesis that mitochondrial respiration contributes to light-evoked hydration changes in the outer retina, with strain differences in respiratory efficiency and imaging responses. This suggests that respiratory-controlled hydration is linked to outer retina expansion and could serve as a biomarker for photoreceptor/RPE complex function, highlighting genetic background as a modifier in retinal disease studies.
研究不足
The study did not randomize the light/dark order due to the time required for dark adaptation. The anesthesia used (urethane for MRI and ketamine/xylazine for OCT) might have effects, though consistency across methods suggests minimal impact. The resolution of MRI methods may limit detection of subtle changes, and the sample size for some measurements (e.g., ADC in 129S6/SvEvTac mice) might be insufficient for statistical power. The water content measured reflects net production minus removal, not isolated contributions.
1:Experimental Design and Method Selection:
The study compared two mouse strains (C57BL/6 and 129S6/SvEvTac) to assess mitochondrial respiration and its link to light-evoked hydration changes in the outer retina. Methods included Seahorse Extracellular Flux Analyzer for OCR and MRC measurements, ultrahigh-resolution OCT for structural imaging, and MRI (proton density and diffusion) for water content and mobility assessment.
2:Sample Selection and Data Sources:
Male 2- to 3-month-old C57BL/6 and 129S6/SvEvTac mice were used, housed under a 12-hour light/dark cycle. Sample sizes were based on previous experience, with specific numbers detailed in figure legends.
3:List of Experimental Equipment and Materials:
Seahorse Extracellular Flux Analyzer, ultrahigh-resolution OCT system (Envisu UHR2200), 7 T MRI system (ClinScan), light source (Mark II Light Source), light meter (Traceable Dual-Range Light Meter), anesthetics (ketamine, xylazine, urethane), and various chemicals (e.g., BAM15, rotenone).
4:Experimental Procedures and Operational Workflow:
For OCR/MRC, retinal punches were assayed ex vivo. For OCT, mice were anesthetized and imaged in light and dark conditions. For MRI, mice were dark-adapted, anesthetized, and imaged with light exposure; proton density and diffusion data were collected and analyzed.
5:Data Analysis Methods:
Statistical analysis used linear mixed models to compare strains and light exposures. OCR data were normalized to cytochrome a content. MRI and OCT data were processed using specific algorithms and in-house code for thickness, water content, and ADC measurements.
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