age-related changes of the retinal pigment epithelium of...

Age-Related Changes of the Retinal PigmentEpithelium of Cats With Chediak-Higashi Syndrome

Linda L. Collier,* Edward J. King,t and David J. Prieur}:

The eyes of 7, 9, and 11-year-old Chediak-Higashi syndrome (CHS)-affected cats were examined bylight, fluorescence, and electron microscopy. Numerous round to oval bodies of various sizes were as-sociated with the retinal pigment epithelium (RPE). These bodies stained positively with periodic acid-Schiflf. They also displayed a bright yellow autofluorescence and stained positively with a prolongedZiehl-Neelsen acid-fast method for demonstration of lipofuscin, suggesting that they contained lipofuscinor a lipofuscin-like material. Ultrastructural examination disclosed the bodies to be secondary lysosomesand large to giant-sized residual bodies. Many of the residual bodies were extracellular and formeddrusen-Iike mounds, covered by deposits of basal lamina, beneath the RPE. Also evident were scattereddegenerated RPE cells and other RPE cells that had detached and migrated into the interphotoreceptorspace. The presence of drusenoid bodies, and the loss of cells from the RPE monolayer in CHS eyeshave not been reported previously. Many of the changes in the CHS cat eyes resemble those in non-CHS aging eyes of man and other species. Invest Ophthalmol Vis Sci 27:702-707, 1986

The Chediak-Higashi syndrome (CHS) is an auto-somal recessive genetic disease of man, cats, and fourother species.1 Enlarged cytoplasmic granules, such assecondary lysosomes and melanosomes, are charac-teristic of the syndrome.2 Hypopigmentation and giantmelanin granules have been observed in CHS humaneyes.3"6 Ultrastructural abnormalities in the retinalpigment epithelium (RPE) of two CHS infants and onechild have been described also.7'8

Hypopigmentation and enlarged melanosomes areevident at the light microscopic level in the eyes ofyoung adult CHS cats.9 The eyes of CHS affected kit-tens are hypopigmented, and giant melanosomes andcomplex granules consisting of melanosomes withinlysosomal matrix are present in the RPE.10 The tape-turn lucidum cellulosum degenerates in CHS kittens."

The effects of longterm abnormality of the lysosomaland melanosomal systems on the RPE of CHS indi-viduals have not been reported previously. In this studywe examined the eyes of aged CHS cats to determinethe morphologic consequences of the CHS defect hav-

From the Department of Veterinary Pathology,*! College of Vet-erinary Medicine, and Department of Ophthalmology,* School ofMedicine, University of Missouri, Columbia, Missouri, and Depart-ment of Veterinary Microbiology and Pathology,^ College of Vet-erinary Medicine, Washington State University, Pullman, Washing-ton.

Supported in part by NIH grants EY03071 and RROO515.Submitted for publication: July 26, 1985.Reprint requests: Linda L. Collier, DVM, PhD, Department of

Veterinary Pathology, College of Veterinary Medicine, University ofMissouri, Columbia, MO 65211.

ing been present over a period of years. Previously un-reported structures included extracellular, autofluores-cent, drusen-like giant residual bodies at the bases ofRPE cells; these bodies were covered by basal lamina.Loss of RPE cells through degeneration of scatteredindividual cells and detachment of others was also ev-ident.

Materials and Methods

The animals were raised and maintained in standardanimal care facilities with cyclic illumination and weretreated in full compliance with the ARVO Resolutionon the Use of Animals in Research. The eyes of CHScats aged 7, 9, and 11 years, and control cats aged 8and 12 years were obtained for microscopic exami-nation. The eyes of the 7- and 11-year-old CHS catswere enucleated and fixed within 1 hr after deaths (sev-eral hours after light onset) of the animals that resultedfrom pneumonia or renal infection. These eyes werefixed in 10% neutral buffered formalin. The other cats(9-year-old CHS, and the 8- and 12-year-old controls)were anesthetized deeply with sodium pentobarbitaland the eyes enucleated 3 hr after onset of morninglight. The right eyes were processed for paraffinembedment for light microscopy and the left eyes wereprocessed for embedment in Epon-Araldite (epoxy)resin for light and electron microscopy.

Light Microscopy

Whole globes were fixed by immersion in Zenker'sfixative or formalin. The lateral calottes were removed

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No. 5 AGING CHANGES OF CHEDIAK-HIGASHI CAT RPE / Collier er ol. 703

from each eye and the remaining central portion wasembedded in paraffin and sectioned at 5 /*m. Each sec-tion was stained with one of the following stains: he-matoxylin and eosin (HE), periodic acid-Schiff (PAS)with and without diastase digestion, or prolongedZiehl-Neelsen acid-fast stain for demonstration of theoxidized lipids present in lipofuscin.12 Additional un-stained sections from each eye were deparaffinized, thenexamined by darkfield fluorescence microscopy usinga mercury lamp with a UG 1 excitation filter havingmaximum transmission at 365 nm and a 450 nm cutofffilter. One-micron thick, toluidine blue stained sectionsof epoxy embedded eyes were also examined by lightmicroscopy. Measurements were made by use of a cal-ibrated ocular micrometer.

Electron Microscopy

The eyes were bisected, the vitreous bodies removed,and the eye cups immersed for at least 4 hr in para-formaldehyde-glutaraldehyde fixative13 at room tem-perature. Vertical strips were cut from the superior edgeto the inferior edge of each eye cup through its center,then subdivided into specimen blocks that wereembedded in Epon-Araldite. The blocks were num-bered to permit comparison of specimens from cor-responding areas of each eye. Ultrathin sections wereexamined and photographed in a (JEOL USA, Inc;Peabody, MA) JEM-100B electron microscope.

Results

Light Microscopy

The appearance of the control cat eyes was typicalof adult cats. The neuroepithelium (with the exceptionof that portion of the RPE overlying the tapetum lu-cidum) and the choroid were uniformly heavily pig-mented with small, dark brown melanin granules.When the unstained control eye sections were illumi-nated by ultraviolet light, a faint, diffuse, yellow au-tofluorescence was visible in the RPE.

The CHS cat eyes were hypopigmented in both theneuroepithelium and the uvea. The melanin presentin the RPE was in the form of giant granules, most ofwhich were located in the peripheral RPE. No melaninwas present in many of the RPE cells in the inferiorportion of the fundus, whereas comparable RPE cellslocated in the nontapetal fundus in normal cats wereheavily pigmented. In the toluidine blue-stained sec-tions of epoxy-embedded tissues, the RPE and cho-roidal melanin granules were various shades of blue,green, gold and brown, rather than the uniform brownobserved in the control eyes.

Numerous pale brown bodies, 5-20 nm in diameter,

at the level of the RPE were evident in the HE-stainedparaffin sections of the CHS eyes. These bodies wereround, oval, or irregular and were more numerous andtended to be larger in the posterior poles of the eyesthan in the peripheral RPE. The bodies stained posi-tively with PAS and prolonged Ziehl-Neelsen histo-chemical stains. With these staining methods, manysmaller positively stained inclusions, ranging in sizefrom 1.25-5 pm, were also evident in the RPE. In un-stained sections of CHS eyes illuminated by ultravioletlight, the RPE bodies displayed a yellow autofluores-cence similar to that characteristic of RPE lipofuscin(Fig. 1A).

In the epoxy sections, numerous round to oval bod-ies, corresponding in size to those observed in the par-affin sections, were present in the RPE. The stainingcharacteristics of the bodies varied greatly, with thecolors ranging from pale blue, almost unstained, todark blue. The appearance of the contents of the bodiesvaried from homogeneous to heterogeneous granular,and melanin granules were incorporated into some ofthe bodies in the peripheral RPE (Figs. IB, C). Thebodies varied greatly in size, with some extending fromthe bases to the apices of, or even distorting the RPEcells (Figs. IB, C). Most of the small to medium-sizedbodies were intracellular inclusions, but some of thelarger bodies elevated the RPE in a manner resemblingdrusen (Fig. 1C). The drusenoid bodies appeared to beextracellular and located on Bruch's membrane, withbasal lamina evident between the inclusions and theRPE cells.

Loss of cells from the RPE monolayer had also oc-curred in the CHS cat eyes. Individual degenerated RPEcells were scattered among cells that were not degen-erated. Many of the degenerated cells were swollen,pale-staining, and most contained enlarged, round, palenuclei (Fig. IB). In addition, widely scattered individualcells (averaging two cells per paraffin embedded section)were present in the interphotoreceptor space. Some ofthese cells may have been macrophages that were pres-ent to remove debris released from disintegrated, de-generated RPE cells, but many appeared to be RPEcells that had detached from the monolayer (Fig. 1D).Since the majority of the CHS RPE cells did not containmelanin, it was not possible to positively identify somecells in the inner retina as migrated RPE cells. In mostof the paraffin sections, two to three heavily pigmentedcells that may have been migrated RPE cells or mac-rophages were present at the levels of the inner plexi-form layer and inner nuclear layer in the peripheralretina adjacent to the ora ciliaris retinae. Greater spac-ing was present between CHS nuclei than control nucleiin the RPE cell layer. In the posterior poles of the CHSeyes, 52 nuclei per 0.5 mm were present, in contrastto 87 nuclei per 0.5 mm in the control eyes.


704 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / Moy 1986 Vol. 27

Fig. 1. The retinal pigment epithelium (RPE) of a 9-year-old CHS cat. A, Autofluorescent giant drusenoid (arrow) and RPE inclusion bodies(arrows). The fluorescent bodies were bright yellow. The autofluorescence of the photoreceptors and choroidal erythrocytes was pale yellow-green, distinctly different from that of the RPE bodies. Paraffin-embedded tissue, unstained, 5-^m thick section (X450). B-D, Epoxy-embeddedtissue, toluidine blue stained, [-fim thick sections. B, A degenerated cell is evident as well as inclusions (arrows) of various sizes and stainingintensities (X1640). C, Drusen-like, giant residual bodies (arrows) beneath the RPE. Material was present in each of the inclusions, but couldnot be demonstrated photographically in some inclusions. An RPE cell with its nucleus was displaced into the interphotoreceptor space (X740).D, Detached RPE cell in the interphotoreceptor space. Inclusions and melanosomes are evident within the cell. The RPE monolayer wasdistorted by a large inclusion (arrow), and smaller inclusions (arrows) are also visible. Bruch's membrane (arrowhead) appears thickened (X1640).

Electron Microscopy

Structures corresponding to the PAS positive, au-tofluorescent bodies of the CHS RPE were large sec-ondary lysosomes and large to giant-sized residualbodies. Some of the largest bodies were actually extra-cellular and surrounded by deposits of basal laminamaterial (Fig. 2 A). The heterogeneous contents of thegiant residual bodies included membrane whorls, ve-

sicular structures resembling remnants of cellular or-ganelles, and discrete particles of material of varyingsizes, shapes, and electron densities. In the peripheralRPE, various forms of melanosomes, many of whichappeared to be degenerated, were also found in thegiant residual bodies.

Each melanin granule that was not incorporated intoa giant residual body was surrounded by a thin layerof secondary lysosomal matrix. A dark, dense layer,

Fig. 2. Electron micrographs of the RPE of a 9-year-old CHS cat. A, A giant residual body had displaced and distorted the apical surface ofthe RPE cell and disrupted the apposition between outer segments and RPE. Note the heterogeneity of the contents of the residual body.Degenerated melanin granules (M) are indicated. Structures that resemble developing melanosomes are indicated by an arrowhead. Also notebasal lamina material (arrows) deposited around the residual body, indicating that the structure is extracellular. Basal membrane infoldings ofthe RPE cells are absent (X6500). B, An RPE cell that is not associated with a giant residual body. Note that each melanin granule is surroundedby a secondary lysosomal matrix. Two large secondary lysosomes (L) are also evident. A discrete deposit of basal lamina is indicated by anarrowhead. Also note the scarcity and small size of the basal infoldings, and the variation in thickness of Bruch's membrane. Phagosomes(arrows) are present in the basal RPE (X 10,500).



B


706 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / May 1986 Vol. 27

often with an irregular surface was present around theperipheries of most melanin granules, just inside thelysosomal layer. The centers of some of the melaningranules were less dense than normal and some had afibrillar appearance and appeared to be degenerated(Fig. 2B).

Alterations in other structures were also observed.Basal infoldings were absent or rare in the CHS RPEcells (Figs. 2A, B). The RPE basal lamina appearedthickened and discrete deposits of material, with theappearance and staining characteristics of new basallamina, were present in many of the RPE cells (Fig.2B). In addition, phagosomes were commonly seen inthe basal portions of many of the RPE cells of the CHScat at 3 hr post light onset (Fig. 2B), in contrast tocontrol cat RPE, in which phagosomes were rarely seenat the same phase of the light cycle.

Discussion

The most obvious abnormal structures of the CHScat RPE were the giant residual bodies. Enormous re-sidual bodies have not been reported previously in CHSeyes, probably because of the relatively young ages ofthe humans and animals studied. The contents of thesestructures in the cats were heterogeneous and appearedto consist of cellular debris, membrane whorls andfragments, and, in the peripheral RPE, melanosomes.Of particular interest were the similarities betweenmany of the CHS giant residual bodies and some ofthe drusenoid bodies observed in non-CHS humaneyes.1415 There were similarities in position of the bod-ies, as well as in the apparent reaction of the RPE cellsin covering the bodies with a thick layer of basal lamina.

Numerous abnormally large inclusions that stainedpositively with PAS and had the ultrastructural ap-pearance of secondary lysosomes were also evident inthe CHS cat RPE. Large secondary lysosomes havealso been reported in the RPE of two CHS children7

and of middle-aged CHS mice (100-200 days old).16

Although discrete granules of the size, shape, and elec-tron density typical of human RPE lipofuscin gran-ules17 were not evident in the CHS cat RPE, the giantresidual bodies and large secondary lysosomes appearedto contain lipofuscin or lipofuscin-like material. Thesestructures autofluoresced similarly to lipofuscin andstained positively by a prolonged Ziehl-Neelsenmethod for the demonstration of lipofuscin. Increasedaccumulations of lipofuscin have also been reportedin the RPE of CHS mice.18 The giant residual bodiesand large secondary lysosomes contained membranewhorls and vesicular membranes, many of whichprobably were derived from phagosomes, whereas oth-ers may have originated as RPE intracellular mem-branes. Degradation of the membranes appeared to

have been impaired. A membrane defect has been re-ported in CHS, involving an increase in unsaturatedfatty acid content.19'20 This could lead to increased per-oxidation of both phagosome-derived and intracellularCHS RPE membranes, making them resistant to ly-sosomal degradation and contributing to acceleratedaccumulation of autofluorescent debris in secondarylysosomes and residual bodies in the CHS RPE.

Loss of melanin from the RPE also occurred withage in the CHS cat eyes. Many of the melanin granulesappeared to be degenerating in the secondary lysosomesand giant residual bodies. Material that appeared to befragments of melanin granules also was present in thegiant residual bodies. Degenerating melanosomeswithin RPE lysosomes have been reported in two CHSchildren as well.7

Also noteworthy, and not reported previously inCHS eyes, was the obvious loss of RPE cells from theRPE monolayer through both death of some cells anddetachment and migration of other RPE cells. Degen-eration and loss of RPE cells also occurs with aging21

and with age-related maculopathies22 in human eyes.In addition to similarities to drusen formation and

lipofuscin accumulation, as well as loss of melanin andRPE cells, there were other changes in the CHS catRPE resembling those in aging non-CHS eyes of manand other species.21"24 These included decreased num-bers and altered structure of basal infoldings, as wellas deposits of discrete mounds of new basal laminamaterial. Thickening of the basal lamina and Bruch'smembrane may also occur in CHS. The CHS cats usedin this study were middle-aged for domestic cats; thus,accelerated aging appears to be a consequence of theCHS basic defect.

Further investigations, including cytochemical andquantitative ultrastructural studies of the eyes of youn-ger adult CHS cats, are currently underway to deter-mine the sources of material in the giant residual bodiesas well as the morphologic sequences involved in de-velopment of the drusenoid bodies and other changesthat occur with aging in CHS eyes.

Key words: Chediak-Higashi syndrome, cats, RPE, aging,drusen, lipofuscin

Acknowledgment

We thank Dr. Lynette Feeney-Burns for helpful adviceand for critical reading of the manuscript.

References

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3. Bedoya V, Grimley PM, and Duque O: Chediak-Higashi syn-drome. Arch Pathol 88:340, 1969.

4. Spencer WH and Hogan MJ: Ocular manifestations of Chediak-Higashi syndrome. Am J Ophthalmol 50:1197, 1960.

5. Johnson DL, Jacobson LW, Toyama R, and Monahan RH: His-topathology of eyes in Chediak-Higashi syndrome. ArchOphthalmol 75:84, 1966.

6. Bregeat P, Dhermy P, and Hamard H: Manifestations oculairesdu syndrome de Chediak Higashi. Arch Ophthalmol (Paris) 26:661, 1966.

7. Libert J, Dhermy P, Van Hoof F, Dufier JL, and Cornu G: Ocularfindings in Chediak-Higashi disease: a light and electron micro-scopic study of two patients. In Genetic Eye Diseases, RetinitisPigmentosa and Other Inherited Eye Disorders, Cotlier E, Mau-menee IH, and Berman ER, editors. March of Dimes Birth De-fects Foundation. Birth Defects: Original Article Series 18(6),1982, pp. 327-344.

8. Valenzuela R and Morningstar WA: The ocular pigmentary dis-turbance of human Chediak-Higashi syndrome. A comparativelight- and electron-microscopic study and review of the literature.Am JClin Pathol 75:591, 1981.

9. Collier LL, Prieur DJ, and King EJ: Ocular melanin pigmentationanomalies in cats, cattle, mink and mice with Chediak-Higashisyndrome: histologic observations. Curr Eye Res 3:1241, 1984.

10. Collier LL, King EJ, and Prieur DJ: Aberrant melanosome de-velopment in the retinal pigmented epithelium of cats with Che-diak-Higashi syndrome. Exp Eye Res 41:305, 1985.

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12. Pearse AGE: Histochemistry: Theoretical and Applied, Vol 2.Baltimore, The Williams and Wilkins Company, 1972, pp. 1076-1085, 1385.

13. Feeney-Burns L and Mixon RN: Development of amelanoticretinal pigment epithelium in eyes with a tapetum lucidum: me-lanosome autophagy and termination of melanogenesis. Dev Biol72:73, 1979.

14. Burns RP and Feeney-Burns L: Clinico-morphologic correlationsof drusen of Bruch's membrane. Trans Am Ophthalmol Soc 78:206, 1980.

15. Farkas TG, Sylvester V, and Archer D: The ultrastructure ofdrusen. Am J Ophthalmol 71:1196, 1971.

16. Robison WG Jr and Kuwabara T: Light-induced alterations ofretinal pigment epithelium in black, albino, and beige mice. ExpEye Res 22:549, 1976.

17. Feeney L: Lipofuscin and melanin of human retinal pigmentepithelium. Invest Ophthalmol Vis Sci 17:583, 1978.

18. Robison WG Jr, Kuwabara T, and Bieri JG: The roles of vitaminE and unsaturated fatty acids in the visual process. Retina 2:263, 1982.

19. Ingraham LM, Burns CP, Boxer LA, Baehner RL, and HaakRA: Fluidity properties and lipid composition of erythrocytemembranes in Chediak-Higashi syndrome. J Cell Biol 89:510,1981.

20. Haak RA, Ingraham LM, Baehner RL, and Boxer LA: Membranefluidity in human and mouse Chediak-Higashi leukocytes. J ClinInvest 64:138, 1979.

21. Sarks SH: Aging and degeneration in the macular region: a clinico-pathological study. Br J Ophthalmol 60:324, 1976.

22. Hogan MJ: Role of the retinal pigment epithelium in maculardisease. Tr Am Acad Ophthalmol Otolaryngol 76:64, 1972.

23. Kornzweig AL: Aging of the retinal pigment epithelium. In TheRetinal Pigment Epithelium, Zinn KM and Marmor MF, editors.Cambridge, Harvard University Press, 1979, pp. 478-495.

24. Katz ML and Robison WG Jr: Age-related changes in the retinalpigment epithelium of pigmented rats. Exp Eye Res 38:137, 1984.


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