Drusenoid maculopathy in rhesus monkeys: autofluorescence, lipofuscin and drusen pathogenesis

Abstract

Purpose: To examine patterns of retinal pigment epithelial autofluorescence and lipofuscin accumulation in relation to drusen and to explore the pathogenesis of drusen in rhesus monkeys.

Methods: The macular areas of six rhesus monkeys, euthanized at 19 to 28 years of age, were studied by bright field and fluorescence light microscopy and transmission electron microscopy.

Results: There was strong autofluorescence in the retinal epithelium that tended to diminish over drusen. Electron microscopy revealed that all retinal epithelial cells had large concentrations of lipofuscin bodies. The epithelial cells overlying drusen, however, tended to have less lipofuscin than epithelial cells not associated with drusen. Electron microscopy revealed that the epithelial cells overlying drusen were losing segments of cytoplasm containing lipofuscin bodies. Macrophage-like cells were consistently present in Bruch's membrane microns away from this lipofuscin-containing cytoplasmic material.

Conclusions: Retinal epithelial cells overlying drusen have less lipofuscin than neighboring epithelial cells. The loss of lipofuscin seems due to a loss of cytoplasm containing lipofuscin that contributes to drusen formation. Macrophages in Bruch's membrane may be responsible for removing this lipofuscin debris. The results support in vivo studies showing reduced autofluorescence over drusen and support the "budding" of epithelial cytoplasm as a source of drusen material.

Figure 1

A. Bright field image of a druse in the macular area of a 28-year-old male monkey: os, is and onl indicate outer segments, inner segments and outer nuclear layer respectively. B. Fluorescent image shows less fluorescence in the epithelium at the dome of the druse (arrow). There is also weaker fluorescence of the inner segments of the photoreceptors, especially cones, and in the choroid‥

Figure 2

includes four sections, each showing the autofluorescence of different drusen in the macular area of a 25-year-old female monkey. The arrows indicate areas of weak or absent fluorescence.

Figure 3

shows a light photomicrograph of a drusen in the macular area of the 25-year-old female monkey of Figure 2. The arrow points out an epithelial cell that has lost much of its pigment.

Figure 4

a-d show electron micrographs of retinal pigment epithelial cells from the macular area of a 26-year-old male monkey, which reveals the enormous amounts of lipofuscin they contain. The arrows in c and d indicate small drusen, too small to detect clinically; ml indicates melanolipofuscin; mi indicates a mitochondria. The calibration at the lower right signifies 2 microns.

Figure 5

shows an electron micrograph of the basal surface of the epithelium and Bruch’s membrane in the macular area of a 19-year-old male monkey. A segment of basal cytoplasm is isolated from the parent layer (arrow); it contains lipofuscin bodies (L). Lipofuscin can also be seen in the intact epithelium; ml, ma and m indicate melanolipofuscin, macrophage-like process; and mitochondrion respectively. The calibration indicates 0.4 microns.

Figure 6

shows an electron micrograph of the basal surface of the epithelium of the 19-year-old male monkey of Figure 5. Here there is also a segment of cytoplasm from a retinal epithelial cell (black arrow) isolated from the parent cell layer and containing lipofuscin (L) and melanolipofuscin (ml). bodies There are also lipofuscin and melanolipofuscin in the intact epithelium. Several smaller segments of cytoplasm are adjacent to the larger arrowed structure; m indicates a mitochondrion among a cluster of these organelles. White arrows indicate macrophage–like processes in Bruch’s membrane. The calibration, lower right, indicates 0.4 microns.

Figure 7

shows electron micrographs of the basal surface of the epithelium from the same monkey of Figure 2 and Figure 3. A. shows a large segment of cytoplasm (d) isolated from the epithelial layer containing many lipofuscin bodies (L). A macrophagic-like process is present (ma). B. shows a magnified view revealing the macrophagic characteristics of structure ma, such as lysosomal bodies, rough endoplasmic reticulum, absence of a basal lamina and iintrusion into Bruch’s membrane where cellular structures are normally absent. The calibration lower right indicates 1 micron for A and 0.4 microns for B.

Figure 8

shows electron micrographs of the basal surface of the epithelium from the same monkey of Figure 2 and Figure 3. This reveals a large segment of epithelial cell cytoplasm containing not only lipofuscin bodies (L) but also atrophic nuclear material (n). There are also several other smaller segments of isolated cytoplasm, one of which also contains a lipofuscin body. Processes of a macrophage-like cell (ma) are adjacent to the degeneration. The calibration, lower right, is 0.5 microns.

Figure 9

shows a druse in the macular area of a 20-year-old male monkey. A shows the druse (D) at low magnification; an oval structure with cytoplasmic-like material is at the center of the druse.(E) indicates an endothelial cell of the choriocappilaris with a basal lamina (arrow); (ma) indicates a macrophage-like process with no basal lamina Magnified views of the left (B) and the right (C) side of the cytoplamic structure show a thickened basal laminar between the intact epithelial layer and the structure but at its outer aspect a basal lamina is absent (arrows). A lipofuscin body (L) is also present in this cytoplasmic structure. The calibration at the lower left indicates 1 micron for A and 0.3 microns for C and D.

Figure 10

shows another druse in this same retina as Figure 9‥A. shows an oval structure with cytoplasmic-like material in the druse but further away from the intact epithelial layer than the similar structure in Figure 9; the epithelial layer is labeled RPE; an endothelial cell of the choriocapillaris is labeled (E) . B shows a magnified view of the cytoplasmic-like structure revealing an absence of any basal lamina. The small process labeled with an asterisk is thought to be a macrophage process; it also lacks a basal lamina. The calibration, lower right, indicates 0.7 microns for A and 0.3 microns for B.

Figure 11

A. shows retinal epithelial cells at the dome of a druse (d) from the macula of a 28-year-old female monkey. The epithelial cells, especially the smaller one on the right, have less cytoplasm and lipofuscin. One cell is pyknotic (thick arrow) and is separating from the epithelial layer which is extremely attenuated at this point (arrows). A magnified view (B) shows a lipofuscin body (arrow) that appears to be is crossing the basal membrane and entering the druse. A small segment of cytoplasmic-like material (asterisk) is present in this drusen. The calibration, lower right, indicates 0.5 microns.

Figure 12

A shows the epithelial layer adjacent to a large druse (B) where there is thinning of the epithelial layer (white arrows) and consequently loss of lipofuscin material in the retinal epithelium near the dome of a druse. The calibration, lower right, indicates 1.2 microns