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Laboratory science
Internal structure consistent with remodelling in very small drusen, revealed by filipin histochemistry for esterified cholesterol Martin Rudolf,1 Katja Seckerdieck,1 Salvatore Grisanti,1 Christine A Curcio2 1
Department of Ophthalmology, University Lübeck, Lübeck, Germany 2 Department of Ophthalmology, University of Alabama at Birmingham, Birmingham, Alabama, USA Correspondence to Dr Martin Rudolf, University Eye Hospital Lübeck, Universitätsklinikum Schleswig-Holstein, RatzeburgerAllee 160, Lübeck 23538, Germany; [email protected] Received 23 August 2013 Revised 17 November 2013 Accepted 2 December 2013 Published Online First 19 February 2014
ABSTRACT Background/aims Drusen, the pathognomonic lesion of age-related macular degeneration, are dynamic and undergo both growth and regression. Using histochemistry to localise esterified cholesterol (EC), we investigated small drusen to discover signs of dynamism. Methods Flat mounts of Bruch’s membrane were prepared from peripheral retinas of six donor eyes without chorioretinal pathology that were preserved within 6 h of death. Tissues were pretreated with ethanol to extract native unesterified cholesterol, incubated with cholesterol esterase and stained with filipin to bind unesterified cholesterol that was newly released by hydrolysis. Tissues were imaged with widefield epifluorescence microscopy. Diameters were measured and internal substructures (shells, lakes) assessed using previous descriptors. Results Of 676 drusen with mean diameter of 26.87 mm, 41.6% were stained homogeneously and 45.7% had lakes of pooled EC. Clusters of 2–7 drusen with similar staining patterns accounted for 25.3% of drusen. Increased EC content near the druse rim (shells) occurred in 10.5%. Conclusions Over half of very small drusen at the edge of clinical detectability have evidence for internal remodelling, suggesting that both formative and removal events are present early in the druse lifecycle.
INTRODUCTION
To cite: Rudolf M, Seckerdieck K, Grisanti S, et al. Br J Ophthalmol 2014;98:698–702. 698
Drusen, the pathognomonic lesions of age-related macular degeneration, are dynamic, exhibiting both growth and regression.1 Large population-based epidemiology studies indicate that eyes with many small drusen eventually exhibit larger and softer drusen with worse prognosis for progression.2 These findings imply expansion and merging at the level of individual lesions. Growth and clustering of drusen over months to several years have been documented by angiography, optical coherence tomography, retro-mode scanning laser ophthalmoscopy and fundus autofluorescence.3–5 Drusen can also regress, fading to spots of geographic atrophy.6 7 A druse at any given moment is balanced between formative and removal processes, which, if ongoing, simultaneously constitute remodelling and may be cyclic.5 While valid systems for experimental study of the drusen lifecycle are still being developed,8 9 histological review of human drusen can supply useful clues to these processes. Lipid is the earliest discovered10 and largest single volumetric component of drusen, accounting for ≥40% of hard druse volume.11 This component
is attributed to an accumulation of apolipoprotein (apo) B and E containing lipoproteins, rich in esterified cholesterol (EC), secreted basolaterally by the retinal pigment epithelium (RPE) into Bruch’s membrane (BrM) for clearance.12 Previously, we, with others, described internal substructures within drusen defined by the relative amounts of esterified and unesterified cholesterol (EC and UC) among other components. One subregion type is a ∼15 mm diameter central core near BrM, originally defined by lectin-binding properties, which are also UC-rich, EC-poor and containing immunoreactivity for non-fibrillar amyloid.13–15 A second subregion type is thin shells at the surfaces of domed drusen that are strongly positive for EC and apo E and C-I immunoreactivity.13 16–18 A third subregion type was polygonal or comma-shaped EC-rich lakes delimited by phospholipid monolayers, which ultrastructurally corresponds to pooled neutral lipid in soft drusen.3 19–21 These regions were distinct from other druse subregions defined by the concentration of ß-amyloid and zinc, and from other particulate components such as melanin, lipofuscin and exosomes.22–25 The size of the smallest drusen detectable in the clinic varies with imaging technology, with base diameters of 30–50 mm reported.4 26–29 Histologically confirmed drusen
Laboratory science
Internal structure consistent with remodelling in very small drusen, revealed by filipin histochemistry for esterified cholesterol Martin Rudolf,1 Katja Seckerdieck,1 Salvatore Grisanti,1 Christine A Curcio2 1
Department of Ophthalmology, University Lübeck, Lübeck, Germany 2 Department of Ophthalmology, University of Alabama at Birmingham, Birmingham, Alabama, USA Correspondence to Dr Martin Rudolf, University Eye Hospital Lübeck, Universitätsklinikum Schleswig-Holstein, RatzeburgerAllee 160, Lübeck 23538, Germany; [email protected] Received 23 August 2013 Revised 17 November 2013 Accepted 2 December 2013 Published Online First 19 February 2014
ABSTRACT Background/aims Drusen, the pathognomonic lesion of age-related macular degeneration, are dynamic and undergo both growth and regression. Using histochemistry to localise esterified cholesterol (EC), we investigated small drusen to discover signs of dynamism. Methods Flat mounts of Bruch’s membrane were prepared from peripheral retinas of six donor eyes without chorioretinal pathology that were preserved within 6 h of death. Tissues were pretreated with ethanol to extract native unesterified cholesterol, incubated with cholesterol esterase and stained with filipin to bind unesterified cholesterol that was newly released by hydrolysis. Tissues were imaged with widefield epifluorescence microscopy. Diameters were measured and internal substructures (shells, lakes) assessed using previous descriptors. Results Of 676 drusen with mean diameter of 26.87 mm, 41.6% were stained homogeneously and 45.7% had lakes of pooled EC. Clusters of 2–7 drusen with similar staining patterns accounted for 25.3% of drusen. Increased EC content near the druse rim (shells) occurred in 10.5%. Conclusions Over half of very small drusen at the edge of clinical detectability have evidence for internal remodelling, suggesting that both formative and removal events are present early in the druse lifecycle.
INTRODUCTION
To cite: Rudolf M, Seckerdieck K, Grisanti S, et al. Br J Ophthalmol 2014;98:698–702. 698
Drusen, the pathognomonic lesions of age-related macular degeneration, are dynamic, exhibiting both growth and regression.1 Large population-based epidemiology studies indicate that eyes with many small drusen eventually exhibit larger and softer drusen with worse prognosis for progression.2 These findings imply expansion and merging at the level of individual lesions. Growth and clustering of drusen over months to several years have been documented by angiography, optical coherence tomography, retro-mode scanning laser ophthalmoscopy and fundus autofluorescence.3–5 Drusen can also regress, fading to spots of geographic atrophy.6 7 A druse at any given moment is balanced between formative and removal processes, which, if ongoing, simultaneously constitute remodelling and may be cyclic.5 While valid systems for experimental study of the drusen lifecycle are still being developed,8 9 histological review of human drusen can supply useful clues to these processes. Lipid is the earliest discovered10 and largest single volumetric component of drusen, accounting for ≥40% of hard druse volume.11 This component
is attributed to an accumulation of apolipoprotein (apo) B and E containing lipoproteins, rich in esterified cholesterol (EC), secreted basolaterally by the retinal pigment epithelium (RPE) into Bruch’s membrane (BrM) for clearance.12 Previously, we, with others, described internal substructures within drusen defined by the relative amounts of esterified and unesterified cholesterol (EC and UC) among other components. One subregion type is a ∼15 mm diameter central core near BrM, originally defined by lectin-binding properties, which are also UC-rich, EC-poor and containing immunoreactivity for non-fibrillar amyloid.13–15 A second subregion type is thin shells at the surfaces of domed drusen that are strongly positive for EC and apo E and C-I immunoreactivity.13 16–18 A third subregion type was polygonal or comma-shaped EC-rich lakes delimited by phospholipid monolayers, which ultrastructurally corresponds to pooled neutral lipid in soft drusen.3 19–21 These regions were distinct from other druse subregions defined by the concentration of ß-amyloid and zinc, and from other particulate components such as melanin, lipofuscin and exosomes.22–25 The size of the smallest drusen detectable in the clinic varies with imaging technology, with base diameters of 30–50 mm reported.4 26–29 Histologically confirmed drusen
