Esterified cholesterol is highly localized to Bruch's membrane, as revealed by lipid histochemistry in wholemounts of human choroid

Abstract

Accumulation of neutral lipids in Bruch's membrane (BrM) is a major age change in human retina and contributes to the formation of extracellular lesions associated with age-related macular degeneration. We developed a BrM-choroid wholemounting technique suitable for reliable staining and evaluated different fluorescent lipid dyes for topographic semiquantitative analysis of BrM lipids. Thin BrM-choroid complexes with partially stripped choroid from 10 aged donor eyes were prepared with an optimized wholemounting technique. Preparation quality was monitored by examining 1-mum-thick sections of representative samples. The staining patterns of Nile Red, BODIPY 493/503, filipin for unesterified cholesterol (UC-F), filipin for esterified cholesterol (EC-F), and Oil Red O in wholemounts were compared with their staining patterns in chorioretinal sections, using wide-field epi-fluorescence microscopy. Wholemounts exhibited optimal flatness on the BrM side. Reduced tissue thickness allowed reliable dye penetration and staining of BrM. Only EC-F was with high specificity localized to BrM and demonstrated an intense and distinct granular staining pattern not previously appreciated in chorioretinal sections. All other lipid dyes also stained choroidal or retinal tissue intensely. No dye provided perfect characteristics in regard to representing all neutral lipid classes present in BrM or to fluorescence intensity. Nevertheless, only EC-F was highly localized to BrM with a specific granular pattern. Because direct assays indicate that esterified cholesterol is abundantly present in BrM, we consider EC-F the most valuable choice for analyzing neutral lipid deposits in human BrM.

Figure 1

Chorioretinal anatomy and preparation quality of Bruch's membrane (BrM) wholemounts. (A) Ten-μm section of the posterior pole of an eye, stained with 1% toluidine-O blue. NR, neuroretina; PR, photoreceptors; RPE, retinal pigment epithelium; Ch, choroid. (B,C) Semi-thin sections of wholemount preparation, here from the macula. Panels show tissue after removal of the retina (top) and removal of large choroidal vessels and most connective tissue (bottom). CC, choriocapillaris. (B) RPE on, including sub-RPE debris and a very small druse (d); intercapillary pillars (black arrowheads) between CC lumens (asterisks); BrM over CC lumens (white arrowheads, called non-pillar areas in wholemount perspective). (C) Sample from a similar region after RPE and sub-RPE debris is completely removed. BrM is intact, and symbols correlate with descriptions in B. Bar = 10 μm.

Figure 2

Wholemount preparation of one donor eye. Asterisk, fovea; optic nerve head is marked by a red scale bead (diameter = 1 mm) in A,C,D. (A) Postmortem central fundus. The retina is blurred with thinned retinal vessels (arrowheads); only the fovea is still transparent. (B) Neuroretina removed. Large choroidal vessels, devoid of blood, appear as lighter lines (arrowheads) in the background. (C) Fundus during RPE removal. Upper-right corner, remaining RPE; choroidal vessels clearly visible. (D) Transillumination of a complete wholemount after removal of all large choroidal vessels, leaving a transparent negative of the vascular network; only choriocapillaris and pigmented connective tissue is left, mounted on a silane coverslip for air-drying. After drying and staining, the coverslip has to be flipped into final position for examination under the microscope.

Figure 3

Neutral lipid stains in sections of retina and choroid. Arrowheads indicate BrM. The RPE is strongly autofluorescent over all investigated spectra, owing to age-related accumulation of lipofuscin. ONL, retina's outer nuclear layer; PR, photoreceptors; Ch, choroid. (A) Unstained section viewed with differential interference contrast (DIC). (B) Section stained with Oil Red O and viewed with bright-field microscopy demonstrates strong red coloration of BrM, including the intercapillary pillars. BrM is slightly detached from the RPE and bowed downward in three locations. (C) Wide-field epifluorescence showing only a very weak fluorescence of Oil Red O. (D) Section stained with Nile Red, demonstrating strong (main) signal in BrM and staining in other structures in Ch and retina as well (strong background signals). The same applies to the BODIPY 493/503–stained section shown in E. (F) Unesterified cholesterol (UC) stained with filipin demonstrated a strong reaction in retina, BrM, and Ch. (G) Isolated signal in BrM found for esterified cholesterol (EC) after pretreatment (see Methods) stained with filipin. (H) Unstained section demonstrating the tissue autofluorescence imaged with the same ultraviolet (UV) filter and image-capture settings as shown in F and G. (I) At higher magnification, EC-F in peripheral section has a granular appearance. Bars: A = 50 μm for A–H; I = 20 μm.

Figure 4

Neutral lipid stains in BrM wholemounts. Outer macula, RPE removed. Arrowheads, intercapillary pillars of BrM; asterisk, BrM over choriocapillaris. (A) In unstained section viewed with DIC, intercapillary pillars were hardly visible. Shown in lower right is remaining choroidal pigment. (B) Section stained with Oil Red O viewed with bright-field microscopy with light-red coloration of BrM and intense outlining of the intercapillary pillars. (C) In dark-field mode, weak fluorescence of Oil Red O of intercapillary pillars only. Wholemounts stained with Nile Red (D) and BODIPY 493/503 (E) show comparable patterns, with strong background fluorescence from the choroid. Intercapillary pillars are visible only if images are regularly enhanced by image-processing software (contrast/brightness). (F) UC stained with filipin. Upper right, an island of RPE was left for better orientation. Weak, inconsistent signal from BrM, major interference by choroidal connective tissue background. (G) EC: filipin regularly stained a granular pattern with increased intensity in intercapillary pillars. (H) Wholemount preparation imaged with same filter (UV) and image-capture settings as in F and G. Bar = 40 μm.