研究目的
To quantify impressions of mitochondrial translocation in degenerating cones and to determine the nature of accumulated material in the subretinal space with apparent inner segment (IS)-like features by examining cone IS ultrastructure.
研究成果
In AMD, macula cones exhibit loss of detectable myoid due to IS shortening in addition to OS loss, as described. Mitochondria shrink and translocate toward the nucleus. As reflectivity sources, translocating mitochondria may be detectable using in vivo imaging to monitor photoreceptor degeneration in retinal disorders. These results improve the knowledge basis for interpreting high-resolution clinical retinal imaging.
研究不足
Transmission electron microscopy and most histological analysis used single sections only. Despite short time between death and preservation compared to most studies using human tissues, mitochondria morphology was not optimal. However, animal models commonly used to study photoreceptor degeneration do not have a cone-rich fovea for color and high-acuity vision or photoreceptors with long Henle fibers typical of human macula. Premortem clinical imaging was not available for the eyes in this study.
1：Experimental Design and Method Selection:
Human donor eyes with advanced age-related macular degeneration (AMD) were screened for outer retinal tubulation (ORT) in macula-wide, high-resolution digital sections. Degenerating cones inside ORT (ORT cones) and outside ORT (non-ORT cones) from AMD eyes and unaffected cones in age-matched control eyes were imaged using transmission electron microscopy.
2：Sample Selection and Data Sources:
Eyes were obtained from the Alabama Eye Bank for research purposes, with a median death-to-preservation time of 2 hours 40 minutes.
3：List of Experimental Equipment and Materials:
Eyes were preserved in 1% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer after anterior segment removal and were stored in this fixative until used. Tissue was postfixed by osmium tannic acid paraphenylenediamine to accentuate extracellular neutral lipids and embedded in epoxy resin (Polybed 812; Polysciences, Warrington, PA, USA).
4：5% glutaraldehyde in 1 M phosphate buffer after anterior segment removal and were stored in this fixative until used. Tissue was postfixed by osmium tannic acid paraphenylenediamine to accentuate extracellular neutral lipids and embedded in epoxy resin (Polybed 812; Polysciences, Warrington, PA, USA). Experimental Procedures and Operational Workflow:
4. Experimental Procedures and Operational Workflow: Macula-wide,
5：8 lm-thick horizontally oriented sections through the foveola and at 2 mm superior to the foveal center (near the rod peak) were stained with toluidine blue. Data Analysis Methods:
The distances of mitochondria to the external limiting membrane (ELM), cone IS length, and cone IS width at the ELM were measured.
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