Tissues were fixed in one-half strength Karnovsky's fixative within four hours of death for a minimum of 24 hours, and transferred to mM sodium cacodylate buffer, pH 7. Tissues from the same eyes processed for electron microscopy were processed for light histological, lectin histochemical, and immunohistochemical studies as described previously [1,59,62].
Ultrastructural examination of donor eyes fixed within relatively short postmortem times reveals five distinct morphological "classes" of drusen in eyes with and without clinically documented AMD.
Loss of pigmentation due to fungal infections
For the purposes of this study, no attempts have been made to correlate these drusen classes to those phenotypes described in the literature.
The purpose of this investigation was simply to examine the composition of all ultrastructural classes of drusen observed in eyes obtained from a large number of donors, representing a significant age range and a variety of AMD phenotypes. One class is manifest by drusen exhibiting a uniformly homogenous substructure. They typically exhibit a rounded to hemisperical morphology similar to what has been described previously as "hard" drusen. However, these results suggest that drusen of this substructural phenotype can be significantly larger than hard drusen described in most clinical grading schemes e.
In addition, drusen with this morphology can be confluent over large areas. A second class is comprised of drusen that are uniformly homogenous at the ultrastructural level of resolution Figure 3Abut in addition to the "granular" material described above, contain small 80 nm diameterelectron lucent, spherical elements Figure 2B. The density of these spherical elements can vary significantly; they are often more dense in the periphery of the drusen.
A distinct subclass of drusen in this category possesses, in addition to the features described above, numerous curvilinear profiles polymerized collagen and larger, spherical elements Figure 3B. Some of the latter are membrane-bounded and all are electron dense Figure 4.
A third class consists of drusen that are highly heterogeneous based on ultrastructural examination Figure 5. Electron dense and electron lucent inclusions, spherical profiles of various diameters, fibrin-like profiles, curvilinear profiles, and, perhaps, cellular debris are present within these drusen Figure 6A.
These drusen are further distinguished by regime prise masse pour ectomorphe calcium-containing inclusions Figure 5 and Figure 6A. These drusen always possess sloped borders are typically wider than they are high, and can exist in confluent layers. A fourth class of drusen is comprised primarily of electron lucent, nm diameter membrane-bounded vesicles Figure 7A. These drusen typically have extremely sloped borders and often exist as a confluent syncitium.
They are most similar in appearance to basal linear deposits that have been described previously. A fifth class of drusen is observed, but only rarely. Drusen of this phenotype contain numerous membrane-bounded vesicles that vary between 80 and nm in diameter Figure 7B. No granular material or other characteristics of the other four drusen phenotypes is observed.
Significantly, no strict relationship between size one important discriminator between "hard" and "soft" drusen class and substructural morphology is noted for four out of the five drusen phenotypes described above. Previous light level histochemical and immunohistochemical investigations using a battery of lectins and antibodies led to the identification of a distinct array of proteins and carbohydrates commonly associated with hard and soft drusen phenotypes as classified at the light microscopic level of resolution [1,59,60].
The studies described below were conducted to determine whether all drusen phenotypes possess this same complement of constituents bound by three representative molecules anti-vitronectin antibody, WGA, and LFA known to react with constituents associated with hard and soft drusen phenotypes. These probes were employed herein to examine the relationship between ultrastructural drusen phenotype and composition.
Examples of drusen labeling are depicted in Figure 8. As anticipated, all three probes bound to all five drusen phenotypes. A number of clinical, histological, and clinicopathological drusen classification schemes have been proposed.
As has been noted [2,15], the difficulties inherent in developing a unified scheme for classification are primarily due to different means of data collection and the resolution of the various techniques employed. In fact, it may be impossible to develop a universal technique that bridges the barrier between fundamentally different means of data collection and analysis.
Furthermore, without reliable information concerning drusen alfalfa pousse cheveux homme, origin, and substructure, classification schemes are inherently limited to gross morphological descriptors. Examination of eyes from over donors fixed within a shorter interval after death than that typically employed in other published studies that possessed drusen reveals five distinct, definable drusen phenotypes at the ultrastructural level of resolution.
Because these substructural phenotypes are observed in drusen of various sizes, shapes, and degree of confluency, we propose that clinical drusen classification schemes that rely on size alone are not a reliable indicator of drusen morphology.
Furthermore, the observations described herein suggest that drusen of all ultrastructural phenotypes may possess a common set of constituent proteins and saccharides, as represented in this study by vitronectin, WGA, and LFA. Additional studies, employing a larger battery of probes to drusen-associated constituents, should help to clarify this suggestion. Why, then, if all drusen are compositionally similar, do hard and soft drusen confer different risks for choroidal neovascularization?
Or, in fact, do they? One explanation is that drusen may be comprised of other proteins, not yet identified, that play a role in determining the pathologic characteristics of these deposits. An alternative explanation is that, even though a number of protein and carbohydrate constituents are common to all classes of drusen, their lipid composition may vary considerably and thus determine their roles in the etiology of AMD.
Indeed, Pauleikhoff and colleagues have suggested that lipid composition may determine the risk posed by sub-RPE deposits . In a somewhat related argument, one cannot exclude the possibility that it is not "hard" or "soft" drusen i.
Rather, it is the composition of drusen that confer this risk. If this proves to be the case, more sophisticated techniques will be required in order to evaluate drusen clinically if one hopes to diagnose various AMD clinical phenotypes.
One approach for determining the full complement of drusen-associated molecules will be to employ more sensitive biochemical assays for detecting the presence of proteins and lipids.
One goal of this laboratory has been to develop a reliable method for collecting drusen-enriched preparations from human donor eyes.
Recent preliminary experiments suggest that enriched drusen preparations are bound by the same probes that bind to drusen in situ, suggesting that these preparations can be obtained without a loss of drusen-associated constituents.
An example of one such enriched preparation is depicted in Figure 9. The observations made in this, and related, studies suggests that, even though a number of ultrastructural classes of drusen can be distinguished, no significant differences in composition between these phenotypes have yet been detected.
Although considerable attention has been paid to clinical and morphological appearance of drusen phenotypes, future studies toward determining the molecular composition of drusen should provide fresh insight into drusen biogenesis and function. As always, a number of significant questions pertaining to the role of drusen in the development of AMD remain.
However, it is likely that significant progress will continue to be made in this field over the next few years. Wasserman Merit Award. We wish to thank the other participants in this session Drs. Richard Green, S. Sarks, J. Sarks, John Marshall, Paulus T. The technical assistance of Ms. Bobbie Schneider, Mr.
Kaj Anderson, Mr. Ihab Ghaly, Mr. Cory Speth, and Ms. Lisa Thayer, as well as the secretarial assistance of Ms. Amy Regan and Ms. Linda Koser are greatly appreciated. Conversations with Mr. Markus Kuehn and Dr. Steve Russell are appreciated.
Drusen retinal pigment epithelium
The authors are especially grateful to Jake Requard, the staff of the Iowa Lions Eye Bank and the all of the tissue donors and their families who gave unselfishly for the advancement of science.
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