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Ebraheim Ismail, Jeffrey Ruberti, Goldis Malek; Evaluation of the morphology of the mouse retinal pigment epithelium/bruch’s membrane/choroid (RPE/BrM/Ch) using quick-freeze, deep-etch transmission electron microscopy (QFDE-TEM) and conventional (cTEM).. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2356.
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The RPE/BrM/Ch is particularly vulnerable in retinal diseases involving the posterior pole such as age-related macular degeneration (AMD), which is the leading cause of vision loss in the elderly in the Western World. Mice are frequently used as a platform to model AMD pathologies because they are easily genetically-modified. QFDE-TEM, a label-free, high-resolution, morphology-preserving imaging method permits the visualization of greater morphological detail than can cTEM. We have previously shown that QFDE-TEM is particularly good at resolving lipid particles in the BrM of human eyes from aged donors. In the present study we ask if QFDE can provide us with important additional information in evaluating the pathology of mouse eyes.
Eyes from aged C57BL/6J mice (control or fed a high-fat diet-HFD) were enucleated and the anterior segments and retinas were removed. In select eyes the RPE layer was also removed. Eyes were fixed in 2% glutaraldehyde. For cTEM: eyes were post-fixed in 1% osmium tetroxide and embedded in Spurrs resin (n=6). Ultrathin sections were stained with uranyl acetate/lead citrate. For QFDE-TEM: tissues (n=17) were “touch” frozen on copper blocks (-196 oC), transferred to a modified Cressington CFE-40 freeze fracture system, fractured, etched and rotary shadowed with Pt/C. Replicas were isolated in 25% bleach solution, washed in DIH2O and imaged on a JEOL JEM1010 TEM.
In the RPE, we catalogued characteristic features in the apical (phagosomes, dense melanosomes), central (cell body, mitochondria) and basal (basal infolding membrane) regions. In BrM, we identified the basement membranes, inner/outer collagenous and elastin layers. Variations in the density of the melanosomes and associated collagen bundles indicated depth in the choroid. In the HFD-fed mice, there were observable differences in baseline morphology due to the presence of lipid particles.
QFDE-TEM enabled the generation of striking, large area mosaic images of the mouse RPE/BrM/CCC complex, complementary and additive to that seen with conventional TEM. These images provide an excellent reference for our ongoing investigations of mouse eyes which demonstrate pathological changes associated with AMD.
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