Insoluble interphotoreceptor matrix domains surround rod photoreceptors in the human retina

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Exp. Eye Res. (1990) 51, 107-110

LETTER

Insoluble

Interphotoreceptor Photoreceptors

TO THE

Matrix Domains Surround in the Human Retina

Photoreceptors in the vertebrate eye project from the outer surface of the retina into an extracellular compartment referred to as the interphotoreceptor matrix (IPM). For many years the IPM was considered only as an unstructured, amorphous entity (RGhlich, 19 70). More recently, studies employing peanut lectin (Johnson, Hageman, and Blanks, 1986 ; Sameshima, Uehara and Ohba, 1987) antibodies to chondroitin sulfate proteoglycans (Hageman and Johnson, 198 7) and cationic dyes which are highly selective for sulfated polyanions (Varner et al., 198 7 ; Hollyfleld et al., 1989) have extended our understanding of the organization of this compartment. Collectively, these studies document the presence of highly organized, relatively insoluble, cone associated subdomains. Within the IPM. each cone photoreceptor is surrounded by a highly organized cylindrical sheath which extends from the outer limiting membrane of the retina beyond the tip of the cone outer segment, terminating at the apical surface of the pigment epithelium. Some evidence suggests that rod photoreceptors may also be surrounded by IPM components that are different from those surrounding cones (Sameshima et al., 1987). However, because retinas used in this study by Sameshima et al, (1987) were flxed immediately after enucleation, it was not possible to establish whether the unique components present around rods were soluble IPM macromolecules that were excluded from regions proximal to cones by the cone matrix domains or whether the rod associated IPM components also represented relatively stable structures, which were equivalent to those investing cones but unique in their chemical composition. Methods have recently been described which permit the isolation of the photoreceptor layer, including the relatively insoluble components of the IPM, from the remainder of human retina (Johnson and Hageman, 1989 ; Hollyfield, Rayborn and Landers, 1990). Using this preparation, we now report that highly organized, interconnected domains are present throughout the IPM surrounding rod photoreceptors. These rod specific domains are resistant to distilled water extraction and can be visualized with fluorescence microscopy following staining with wheat germ agglutinin (WGA) conjugated to rhodamine. Human donor eyes, with post-mortem times of under 1 hr. were obtained from the Lions Eyes of Texas Eye Bank. Houston, TX. Following procedures described previously which involve placing unfixed, 00144835/90/070107+04

%03.00/O

EDITORS

Rod

freshly isolated retinal fragments in distilled water (Hollyfield et al., 1990), the photoreceptor layer including the insoluble components present in the IPM were separated from the inner retina. While in distilled water, the isolated outer retina swells to approximately twice the dimensions of the original retinal fragment from which it was derived. This differential swelling of the IPM/photoreceptor complex relative to the swelling of the inner retina may be in part responsible for the separation of these two components. Following detachment of the two layers, the nearly transparent, greatly expanded outer retina/IPM fragments are retained for further analysis while the opaque fragments, consisting of inner retinal layers, are removed with forceps and discarded. In distilled water, the outer retina/EPM isolates are easily manipulated with forceps or moved from one dish to another with a pipette. However, when placed in Ringer’s or PBS, the isolates shrink to the original dimension of the retinal fragment from which they were derived and become extremely sticky and virtually impossible to manipulate. To avoid the difficulties of manipulating sticky isolates during processing for fluorescent microscopy, fixation and subsequent rinses were carried out using distilled water in the absence of salts or buffer. Fixation of the isolates was with 4% formaldehyde in distilled water for 30 min to overnight. After several distilled water rinses to remove excess formaldehyde, isolates were placed in 3 S-mm plastic Petri dishes and were oriented with the sclerad surface either toward or away from the Petri dish surface. This orientation is relatively easy to establish since the fragments tend to curl and form tubular- to cap-shaped structures. The sclerad surface of the isolate is always located on the convex side. When appropriately orientated, the isolates are forced to the dish bottom and gently tacked in place by pressing the isolate to the plastic surface at two or three positions with the point of the forceps. Approximately one-half of the distilled water is then gently pipetted from the dish and replaced with PBS. The addition of PBS immediately causes the isolate to shrink to the original dimension and the increased adhesiveness in the presence of salts causes the isolate to adhere firmly to the bottom of the plastic dish. This adhesiveness to the plastic is very helpful in maintaining the orientation and flattened contour of the specimen during all the subsequent processing steps. Withdrawal of fluid and addition of PBS is repeated three to four times as gently as possible. It is also 0 1990 Academic Press Limited

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J. G. HOLLYFIELD

ET AL.

FIG. 1. Isolated IPM complex from the human retina stained with WGA-rhodamine and viewed from the level of the outer limiting membrane in a sclerad direction. Fluorescence is present associated with a complex network surrounding rod photoreceptors. Note the circular openings through the matrix at the level of the outer limiting membrane. Lightly fluorescing cone photoreceptor domains are also present (asterisk). FIG. 2. Isolated IPM complex from the human retina stained with WGA-rhodamine and viewed from the level of the pigment epithelium in a vitread direction. The dark, circular recesses represent cone photoreceptor domains which only faintly stain with this lectin (asterisk). The intense fluorescence of the tubular profiles throughout the remainder of the micrograph represents insoluble WGA-rhodamine staining components in the IPM which surround rod photoreceptors. A circular opening into the tip of many of these cylindrical compartments is evident (arrows). Note the close lateral association of the individual rod domains, suggesting lateral interactions among all the individual units. Figures I and 2 were photographed and printed at the same magnification. Bar in lower right of Fig. 2 represents 10 Lirn. FIG. 3. Double exposure micrograph of the isolated IPM complex stained with both FITC-labeled PNA and rhodamine-labeled WGA and viewed from the level of the outer limiting membrane in a sclerad direction (the level of focus is slightly more sclerad and at a more oblique angle than employed for Fig. 1). The larger, intensely fluorescing circular to oval profiles represent the base of the cone matrix domains at their point of origin at the level of the outer limiting membrane (in color these fluoresce a bright green). In the surrounding areas, the rod matrix domains form an interconnected honeycomb pattern (in color these components fluoresce red). FIG. 4. Rhodamine fluorescence of the same area presented in Fig. 3 above. In this single fluorescent image, the

IPM

DOMAINS

SURROUND

ROD

PHOTORECEPTORS

extremely important during all these steps not to let the isolate interact with the air/liquid interface or it quickly spreads onto the aqueous surface and disintegrates. The IPM isolates were stained either with rhodamine-conjugated wheat germ agglutinin (WGA) (Sigma Chemical Co., St Louis, MO) at a concentration of 200 ,ug ml-* for 20 min or a mixture of rhodamineconjugated WGA and FITC-conjugated peanut lectin (Vector Labs, Burlingame, CA), each at a concentration of 200 ,ug ml-’ for 20 min. After three to four 10 min rinses with PBS, a 22-mm2 cover slip was floated on the surface of the PBS and then gently lowered into close proximity of the dish bottom by carefully removing fluid from the dish margin with a pipette. The Petri dish was then attached to a microscope slide with a small piece of double stick tape. This assembly was then placed on the stage of a Zeiss Axiophot photomicroscope equipped with epifluorescent illumination for viewing and photography. Some of the isolates were incubated with WGArhodamine in the presence of 0.2 M N-acetylglucosamine. The WGA-rhodamine fluorescence described below was greatly reduced by this sugar. WGA-rhodamine intensely labels the isolated IPM complex in a pattern consistent with the presence of a highly organized, insoluble domain associated with rod photoreceptors. When viewed from the level of the outer limiting membrane in a sclerad direction, the WGA-positive IPM components appear as a highly organized honeycomb-like structure with circular openings into which project the inner segments of the rod photoreceptors from the outer retinal surface. Brightly fluorescing lines, suggesting filamentous subcomponents appear to extend throughout this three-dimensional network (Fig. 1). The matrix components surrounding the rods are highly interconnected with each other and do not appear as distinct individual compartments. Their morphology gives the appearance of a highly interacting unit which occupies the entire extracellular compartment surrounding the rod photoreceptors. The only truly isolated compartments are the cylindrical openings into which the individual rod photoreceptors project from the outer retinal surface. In the peripheral retinal samples from which all these isolates were taken, the cone matrix domains are only lightly fluorescent with WGArhodamine and appear as dark recesses in the highly organized, brightly fluorescing fields of rod associated matrix components. In IPM samples viewed from the level of the pigment epithelium, downward onto the IPM isolates, the WGA-rhodamine positive rod associated compartments retain their cylindrical shape and appear to be

109

closely associated with one another through lateral interactions of these IPM components (Fig. 2). At the tip of each rod compartment, a small circular opening is present. These openings are presumed to be at the level where the IPM would abut onto the apical surface of the pigment epithelium prior to retinal isolation. The cone matrix domains only faintly stain with WGA-rhodamine, allowing their tubular luminal openings to be just discernible from the domain wall. A close association between the rod associated IPM and the cone matrix domains is suggested by the apparent close associations between these two components when double exposure micrographs are taken of the double stained isolates (Figs 3 and 4). The overall appearance of the IPM when both rod and cone matrix domains are imaged in these double stained preparations is that the matrix domains surrounding both photoreceptor types are closely associated with each other allowing the IPM to function structurally as a single unit rather than separate matrix domains which lack lateral interaction. In several instances, in regions where the IPM isolate was tightly adherent to the substratum of the Petri dish, when distilled water was replaced with PBS, the isolates contracted and were stretched between two or more points of attachment. When this occurred, all the individual matrix domains were deformed in the same pattern, suggesting that extensive lateral interactions bind the individual rod and cone matrix domains together to form a highly organized and adherent IPM compartment (not shown). The failure of these IPM components to be solubilized in distilled water suggests that at least a portion of the extracellular matrix surrounding both rod and cone photoreceptors is relatively stable. The structural integrity of the matrix, along with the suggested lateral interactions between the individual domains, suggests that these components confer on the IPM a mechanical integrity, allowing it to behave as a highly organized structural unit containing cylindrical openings through which the rod and cone photoreceptors project. A mechanically stable matrix, which defines the shape of the IPM, may be in part responsible for aligning and restricting the orientation of the entire population of photoreceptor outer segments to the appropriate optical axis of the eye. The turnover of these IPM components, their development and cellular origin, as well as their functional role, will be the subject of additional studies. While our most extensive analysis of these rod associated domains has been made on human tissues, we have preliminary observations which indicate that similar, relatively insoluble rod associated extracellular domains are also present in mouse, cat, dog, rabbit,

interconnected appearance of the WGA-rhodamine stained components surrounding rods is more apparent. Note that the IPM components surrounding cones appear as pit-lie depressions with sloping shoutders in this single exposure image. See text for further explanations. Figures 3 and 4 were photographed and printed at the same magnification. Bar in lower right of Fig. 4 represents 10 ym.

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bovine and sheep retinas. A more complete description of the regional variations in the subdomains of the IPM in the human retina, as well as variations of these components in several species, will be dealt with in future reports.

Acknowledgements We thank Mr Alex Kogan and Mrs Gilma Miranda for help with the photography and the Lions’ Eyes of Texas Eye Bank,

Houston, for their efforts in obtaining the human tissues used in this study. This study was supported by grants from the National Eye Institute, National Institutes of Health, Bethesda, MD, The RP Foundation Fighting Blindness, Inc.. Baltimore, MD, The Retina Research Foundation, Houston, TX, and Research to Prevent Blindness, New York, NY. Some of the instrumentation used in this study was obtained through funding by The Institute for Aid and Rehabilitation, Houston, TX. J.G.H. is the recipient of an Alcon Research Institute Award and a Research to Prevent Blindness Senior Scientific Investigator Award. JOE G. HOLLYFIELD” MARY E. RAYBORN ROBERT A. LANDERS KATHY M. MYERS

Cullen Eye Institute, Baylor College of Medicine, Houston, TX 77030, U.S.A. (Received *

For

ET AL.

22 November

1989 and accepted

27 November

1989)

reprint requests.

References Hageman, G. S. and Johnson, L. V. (1987). Chondroitin 6sulfate glycosaminoglycan is a major constituent of primate cone photoreceptor matrix sheaths. Cum. Eye Res. 6, 63946.

Hollyfield,J. G.,Rayborn, M. E.andLanders,R. A. (1990). A techniquefor isolationof the photoreceptorlayer from other neuronsin the human retina. Exp. Eye Res. 50. 335-8. Hollyfield. J. G., Varner, H. H., Rayborn, M. E.and Osterfeld, A. M. (1989). Retinal attachment to the pigment epithelium: Linkage through an extracellular sheath surroundingcone photoreceptors.Retina 9, 59-68. Johnson,L. V. andHageman,G. S.(1989). Characterization of isolated cone matrix sheath substructure. Invest. Ophthalmol. Vis. Sci. (Suppl.),490.

Johnson,L. V., Hageman, G.S. and Blanks, J. C. (1989). Interphotoreceptormatrix domainsensheathvertebrate conephotoreceptorcells.Invest. Ophthalmol.Vis. Sci. 27, 129-35.

Rijhlich, P. (1970). The interphotoreceptormatrix : electron microscopic and histochemicalobservationson the vertebrate retina. Exp. Eye Res.10, 80-96. Sameshima,M., Uehara,F. and Ohba, N. (1987). Specialization of the interphotoreceptormatricesaround cone and rod photoreceptorcellsin the monkey retina, as revealed by lectin cytochemistry. Exp. Eye Res. 45, 845-63. Varner, H. H., Rayborn. M. E., Osterfeld,A. M. and Hollyfield, J. G. (1987). Localizationof proteoglycan within the matrix sheathof conephotoreceptors.Exp.Eye Res. 44, 633-42.