convolution in human rods: an ageing process

British Journal of Ophthalmology, 1979, 63, 181-187

Convolution in human rods: an ageing processJOHN MARSHALL,' JOHN GRINDLE,2 PAUL L. ANSELL,1 ANDBESSIE BORWEIN3From the 'Department of Visual Science, Institute of Ophthalmology, London, England; the 2Department ofClinical Ophthalmology, Institute of Ophthalmology and Moorfields Eye Hospital, London, England; andthe 3Department of Physics, University of Western Ontario, London, Canada

SUMMARY In a morphological survey of 73 human retinae spanning 9 decades, and including 20retinae which were obtained from eyes enucleated for malignant melanomas, nodular excrescenceswere noted in the outer segments of rods with an incidence which increased with age. Thesestructures were sectioned in both their vertical and horizontal axes and on electron microscopicalexamination were seen to result from the localised convolution of affected outer segments. Thetopographic morphology of such convolutions is described and their modes of formation arediscussed.

The process of ageing and its manifestations in thecells of the human retina has for some time been amajor research interest in our laboratory. Thesestudies include the electron microscopical examina-tion of 73 human retinae obtained from bothenucleations and cadaver eyes. This series containsspecimens from each of 9 decades, the youngest eyebeing 19 years and the oldest 90 years. The detailedmorphology of the photoreceptor cells, the pigmentepithelium, and Briich's membrane have beeninvestigated in order to establish control referencematerial for a future study of senile maculardegenerations and inherited retinal dystrophies. Wewere interested therefore by a recent report pur-porting to show the fusion of the outer segments ofjuxtapositioned rod photoreceptor cells in the retinaof a 47-year-old woman whose eye was enucleatedbecause of the presence of a choroidal melanoma(Borwein et al., 1977). In the present paper morpho-logical evidence shows that this suggested fusion ofappositioned cells is an age-related process occurringwithin the outer segments of individual rods.

Methods

One of the commonest conditions that leads toenucleation of human eyes is malignant melanomaof the uveal tract. The retinae of 20 such eyes havebeen studied in the present survey, with an age

Address for reprints: Dr John Marshall, Department ofVisual Science, Institute of Ophthalmology, Judd Street,London WC1H 9QS

range spanning 7 decades. The immediate post-operative procedures varied with the individualdemands of participating centres. In most centrescorneal discs were trephined for subsequent use ingraft procedures. One centre required the dissectionof the globe and the hemisection of the unfixedtumour so that samples could be processed for tissueculture as well as histopathology. In some casesintact globes were presented immediately afterenucleation, and here transcorneal incisions weremade in the sagittal plain before immersion infixative.

All eyes were fixed within minutes of enucleationby immersion in 100 ml of 0 3 M glutaraldehydebuffered in 0-1 M sodium cacodylate containing10 mg/ml of calcium chloride with a final pH 7-4.Intact globes were progressively dissected in thissolution, the cornea being removed at the sulcussclerae after 5 minutes, the iris after a further 5minutes, and the lens, ciliary body, and vitreous 20minutes after the initial immersion.

After a total of 1 hour in fixative all posterior eyecups were washed in 0-1 M sodium cacodylatecontaining 7-5% sucrose, and in this solutionexperimental samples were isolated under a dis-secting microscope. In all cases the tumour wasisolated first and subsequently processed forconventional histopathology. Where the topographyof the tumour allowed, 5 sets of samples wereisolated for electron microscopical study. Thesewere the macula and 4 bands of tissue radiatingfrom the macula to the ora serrata in the cardinalaxes. All samples were dissected so that their

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orientation with respect to the macula could bedetermined during subsequent ultramicrotomy, andtheir distance from this locus was recorded. Theisolated specimens were postfixed for 1 hour in 2%osmium tetroxide in 0-2 M sodium cacodylate beforebeing dehydrated by progressively increasing con-centrations of ethanol in water and embedded inEpon 812 via epoxypropane.

Cadaver eyes were processed as above, beingprogressively dissected in glutaraldehyde. Wherepossible, the time interval between death andenucleation was recorded, as was the time betweenenucleation and fixation.

All sample blocks were first mounted in aCambridge Huxley ultramicrotome and 0-8-V±msections were cut with glass knives. Most blockswere cut in at least two planes, first along the axisof the photoreceptor outer segments, and secondlyat right-angles to this axis. Thick sections werestained with either toluidine blue (Meek, 1963) orbasic fuchsin and methylene blue (Huber et al.,1968). By means of a chuck adaptor orientatedblocks were transferred to a Reichert OMU3ultramicrotome where 400-A (40-nm) sections werecut with a diamond knife. These sections forelectron microscopy were mounted on 200-meshcopper grids and stained with uranyl acetate andlead citrate before being examined in an AEI 801electron microscope.

Further samples, representative of each decade,were isolated after glutaraldehyde fixation, andthese were frozen in isopentane precooled in liquidnitrogen before being mounted in a Slee cryostat.Frozen sections 10,m thick were examined undera Zeiss microscope fitted with an epifluorescenceattachment to determine the relative autofluorescencedue to lipofuscin in the retinal pigment epithelium.

Results

Extensive observations in the present study haveshown that in eyes enucleated for choroidalmelanoma the morphologies of areas of retinaeremote from the tumour are indistingushable fromcomparable areas of retinae in eyes of similar ageobtained from cadavers with no known ophthalmicdisease. These observations are in agreement withprevious studies (Borwein et al., 1977). In mostcases the fixation of retinae in surgically enucleatedeyes was superior to that achieved in post-mortemspecimens. However, significant observations weremade on both groups and statistical data werepooled.When transverse sections of retinae in the second

and third decades were examined under the lightmicroscope, the outer segments of rod photoreceptor

cells were seen as homogeneously staining elongatedrectangular systems with regular parallel borders intheir long axis. In samples from the central or peri-macular regions such outer segments were approxi-mately 1-4 ,um in diameter and between 24 and 27 ,umin length (Fig. 1). The discrete nature of thesesystems was apparent in sections cut along the axisof the outer segment and in those at right-angles tothis axis, as in both cases a poorly stained inter-receptor matrix was distinguished. In all sectionsthe rod outer segments experienced an angulardeviation between their ciliary region and the pigmentepithelium, and the degree of inclination varied withretinal position, a finding common in other verte-brates (Laties and Enoch, 1977).Changes in the morphology of rod outer segments

began to appear in the fourth decade and occurredinitially in the perimacular regions. In light micro-scopic sections such changes were seen as nodularswellings, distorting either the central region, orpigment epithelial third, of the rod outer segmentprofile (Fig. 1). The rod diameters were increased tobetween 2-5 and 3-5 ,um in the region of thesedistortions. With increasing age such distortionsbecame apparent in all areas of retina and in anincreasing number of rod cells. By the seventhdecade between 10 and 20% of the total rodpopulation was involved.

In the electron microscope, sections of rod outersegments of young eyes showed few deviations fromthe typical ordered membrane array presented bythe stacks of lobed discs. When such deviationsoccurred, they usually presented as groups of 10 to30 discs which had become disorientated with respectto both their fellows and the boundary membraneofthe cell. In some instances several such dislocationsof the disc array occurred within the outer segmentof a single receptor cell. A further anomaly was theoccasional presence of regions of small vesicleswhich occurred parallel to and within the plane ofthe disc membranes. One or both of these phenomenaoccurred in some receptor cells in every sampleexamined. However, they never involved theboundary membrane of the cell, whose parallelconfiguration was always preserved in these regions.Small dislocations of the disc stacks which involvedthe boundary membranes were occasionally observedin young eyes, but these were always at the tips ofrods, where they were surrounded by the receptorsheaths of the pigment epithelium. These occur-rences usually resulted from the presence of dis-organised or shed discs prior to their beingphagocytosed.

In the rods of the older eyes the nodular expan-sions were identified as areas in which the outersegments had corrugated within their long axes

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Convolution in human rods: an ageing process

Fig. 1 (a) Photomicrograph of atransverse section showing thereceptor epithelial junction in theperimacular region of the retinaof a 19-year-old male. Theboundary membranes of the rodsare parallel with no excrescences.Bruch's membrane is thin and thepigment epithelium contains littlelipofuscin.

(b) Photomicrograph ofatangential section through thereceptor outer segments in theretina seen in (a) and demonstrat-ing the isolation of appositionedelements.

(c) Photomicrograph of atransverse section through thereceptor epithelial junction in theperimacular region of the retinaof a 54-year-old female. Nodularexcrescences can be seenassociated with several rod outersegments. Bruch's membrane isthicker than in (a) and an increasedlipofuscin content is apparent.

(d) Photomicrograph of atangential section through thenodular region of the outersegments shown in (c). Severalexamples of very closelyappositioned elements aredemonstrated (arrowed). The barmarker is 10 Fm

(Fig. 2). These corrugations occurred in two forms.In some rods the disc stack was displaced in a sinu-soidal curve, which was compressed within the cells'boundary membrane. In other rods the whole outersegment including the boundary membrane wasdistorted into a series of curves. Both configurationsresulted in localised corrugations in which themembranes in the ascending and descending limbswere closely appositioned. These convolutionsincreased the absolute length of the outer segmentsby 6 to 10 [±m and their disc content by 20 to 40%.However, the axial distance between their ciliumand the pigment epithelium remained in the samerange as those of young eyes. These systems werenever found to extend any distance into the receptorsheath systems of the apical membrane of the pig-ment epithelium. Within the sheaths small disloca-tions of the disc stacks were occasionally observed,

and these were similar to those seen in young eyes.When the convolutions were sectioned at right-

angles to the long axis of the outer segments theyappeared as a fused association of independentelements from several juxtapositioned cells (Fig. 2).The number of elements involved depended uponthe number of convolutions experienced by aparticular outer segment but ranged between 2 and 5discs. The 2 modes of outer segment corrugationwere clearly distinguishable, with groups of discseither being bounded by a single membrane system,or multiple boundary membranes with varyingdegrees of confluence. These sections also demon-strated that multiple convolutions were not confinedto a single plane (Fig. 2). Similar changes have alsobeen observed in the rod outer segments of a mature(10 to 15 years) rhesus monkey.

In human retinae in the present study inter-

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a

AxA..

.4i1.

B'Fig. 2 (a) Electron micrographof a transverse section through arod outer segment nodule showinga convolution of the disc stackand varying degrees of membranefusion between the ascending anddescending limbs. Therefore atangential section on plane A-A'would appear as a system ofdiscs enclosed by a single boundarymembrane as in (b), while asection on plane B-B' wouldappear as 3 discs each with theirown boundary membranes. Thebar marker is I t±m

(b) An electron micrograph ofa tangential section through a rodouter segment nodule as describedin (a). The bar marker is I l±m

receptor membrane associations were found betweenprocesses from the inner segments (Fig. 3). Thesewere similar to those previously described in otheranimals (Fain et al., 1976). However, in all suchassociations the integrity of participating membraneswas maintained, and no fusion of cells was observed.A detailed account of the changes observed in the

pigment epithelium and Bruch's membrane in thepresent survey forms the basis of a future report.However, certain of these changes may be pertinentto those seen in the rods and are therefore brieflymentioned. In all retinae examined electron-dense

deposits of lipofuscin were observed within thepigment epithelial cells, and both electron andfluorescence microscopy confirmed previous findingsthat such deposits increase with age (Hogan et al.,1971). In young eyes lipofuscin inclusions were mostabundant in epithelial cells in the macular andperimacular regions.

Discussion

Loss of order in the outer segments of photo-receptor cells occurs in response to both pathological

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Fig. 3 (a) Low-power electronmicrograph of a tangential sectionthrough the inner segments of thephotoreceptor cells in a 54-year-old woman. The interreceptormatrix is much reduced in thisregion of the retina but stillapparent. The bar marker is 2 s±m.

(b) Electron micrograph of asimilar section to that in (a) takenjust external to the outer limitingmembrane. Specialised junctionalcomplexes may be seen bothbetween appositioned rods(closed arrows) and between rodsand cones (open arrows). The barmarker is I tim

and histological processes. In vivo the receptorsrespond to a variety of disturbances in their micro-environment by failing to maintain the regularperiodicity of their component disc membranes.After mild or transient injuries angular translocationsof groups of discs occur within the disc stack. Inepisodes of severe or protracted interference withreceptor homoeostasis the disc membranes degener-ate into vesicular or whorl-like configurations.

Typical examples of these degenerative changes areseen in such conditions as retinal detachment (Krolland Machemer, 1968), vitamin deficiencies (Hayes,1974), and thermal (Marshall, 1970a) or photicinjury (Kuwabara, 1970). In each of these conditionsthe persistent changes in outer segment morphologyoccur as a result of fusion of membranes of bothindividual and juxtapositinined discs. This capacityfor disc fusion is also seen in an extracellular

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environment and between discs originating fromdifferent receptor cells, as in the RCS rat discsshed from the rod tips, which are not phagocytosedby the pigment epithelium but come together toform huge sheets of lamellar material (Dowling andSidman, 1962). Similar examples of disc membranefusion are seen in vitro in experiments in whichisolated rod outer segments are exposed to osmoticshock (Falk and Fatt, 1973) and in some histologicaltechniques in which certain types of fixative inducesuch membrane fusion (Tormey, 1964). Theseobservations imply that active processes are requiredfor the continued maintenance of disc integrity. Incontrast to disc membranes the boundary membranesof the outer segments of receptor cells remaindiscrete throughout each of these pathologicalprocesses and never fuse with those of neighbouringcells. The one exception to this is that portion ofthe boundary membrane shed from the tips of rodsin the RCS rat.

In the present study areas of disorganisationarising from degenerative processes involving thediscs were few in young eyes and consisted of theangular translocation of groups of discs within theboundary membranes of the rod outer segments.The vesicles seen within the plane of the discsresulted from section planes running either throughthe disc lobes or crenulations or through thefimbriae associated with the ciliary incisure (Pedlerand Tilly, 1967). These latter incursions in the discmembrane do not maintain their stacking registeron passing away from the cilium and usuallyrevolve varying degrees in their axis of symmetry ontheir passage down the outer segment. Thus isolatedor single multiple rows of vesicles with axial dimen-sions identical to those of the discs are observedthroughout the length of the outer segments.Changes in the appearance of receptor outer

segments induced by histological processing are wellknown, and in all ophthalmic pathology laboratoriesthe maintenance of the correct stereo spatialrelationships between the neural retina and the pig-ment epithelium is a perennial problem. Manyhistological techniques result either in the detachmentof the retina or in its compression against thisunderlying layer. In this latter group the receptorouter segments are either distorted to run at anacute angle towards the pigment epithelium, or theyassume a sigmoid appearance. In affected samplesall the receptors in any given area are similarlydistorted. The convolutions that occur in the outersegments of older eyes in the present study (Fig. 2)are not an artefact resulting from the tissue process-ing techniques. They show a discontinuous distribu-tion in histological sections of retina and an increas-ing incidence with age.

In many animals (Young, 1976) including monkey(Young, 1971), autoradiographic studies have shownthat the discs in rod outer segments are in a state offlux. These experiments show that throughout lifenew discs are continuously formed in the region ofthe cilium, and are progressively displaced down theouter segment towards the pigment epithelium bysuccessive disc production.

In monkeys the outer segment transit time for adisc is 9 to 13 days (Young, 1971). Old discs at thetips of the outer segments are removed by thephagocytic action of the pigment epithelium, aprocess which exists in dynamic equilibrium withthat of disc production. Once inside the pigmentepithelium the groups of engulfed discs, calledphagosomes, undergo lysis, which induces theirprogressive degeneration (Marshall, 1970b; Ishikawaand Yamada, 1970). The degenerative end productsof lysis are voided from the epithelial cells viaBriich's membrane into the choriocapillaris. Fromsimple calculations it can be shown that in themonkey each epithelial cell destroys well over 1000discs each day. This amounts to an enormousquantity of membranous material passing throughthe pigment epithelium during the life span of ananimal.

This system has not been studied autoradiogra-phically on human material, but substantivemorphological evidence indicates its existence(Spitznas and Hogan, 1970). Morphological studiesalso suggest that the pigment epithelium in man isinvolved in the phagocytosis of cone outer segments(Steinberg et al., 1977). The observations of increas-ing deposits of lipofuscin within the pigmentepithelium coupled with a progressive build-up ofinclusions in Briich's membrane in ageing humaneyes have been interpreted as the products of in-complete breakdown or transport of such phago-cytosed outer segment material (Hogan, 1972).

It has also been suggested that both of theseinclusions are manifestations of a reduction in theability of the pigment epithelium to contend with itsphagocytic load and that their presence furtherdisturbs the dynamic equilibrium of rod growth andphagocytosis. Such perturbations could arise from aprogressively increasing impedance to the outflow ofphagosomal debris induced by the successive deposi-tion of such material within Briich's membrane. Thismechanism could therefore result either in a massivebuild-up of debris, with the production of drusenand possibly more serious senile lesions (Sarks,1976) or in a decrease in the rate of phagocytosis.

If the rate of disc production remains relativelyconstant throughout life but the rate of phagocytosisdecreases, then rods must increase in length. Suchan imbalance could well result in the convolutions

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described in the present study. This suggestion issupported by two further findings. First, the initialoccurrence and subsequent highest incidence of theseconvolutions is in the perimacula, an area in whichretinal pigment epithelial cells have a large lipofuscincontent, and secondly this is an area in which drusenare commonly observed (Gass, 1973).The degree of membrane fusion between the

various limbs of the convolutions could result inthree different appearances in sequential cross-sections of a single convolution (Fig. 2). Thus,where the boundary membranes of the limbs hadnot fused, 'isolated' elements were observed; wherethese membranes were confluent, the systemappeared as 2 or more discs within a single mem-brane. And, finally, various intermediate fusionsimparted an appearance similar to cytoplasmicbridges. This latter form resulted in the mis-interpretation of these systems as the fused elementsof appositioned receptor cells (Borwein et al., 1977).The present findings are a reinterpretation arisingfrom examination of data in both laboratories.The functional implications of convoluted rods

and the effects of their increasing numbers withage are unclear. The increase in disc content andabsolute outer segment length does not seemsignificant because their axial length remainsrelatively constant through life. The increase in sizeof the catchment area presented to incident light inaffected receptors may be of greater consequence torod function. However, the variations betweenindividual receptors would render analysis extremelydifficult.

It is our pleasure to thank the medical and nursing staffs ofthe following hospitals whose co-operation in gatheringsamples has been essential: Addenbrookes Hospital,Cambridge; Hillingdon Hospital, London; King's CollegeHospital, London; Manchester Royal Eye Hospital;Middlesex Hospital, London; Moorfields Eye Hospital,London; and Victoria Hospital, London, Ontario. We aregrateful to Miss E. Clarke and Mrs A. Karkahanis fortechnical assistance, and to both the Garrick CharitableSettlement and the Wellcome Trust for donations ofscientific apparatus. We also acknowledge the US ArmyMedical Research and Development Command, and theAdvisory Group for Aerospace Research and Development(AGARD) of NATO, for financial support.

References

Borwein, B., Medeiros, J. A., and McGowan, J. W. (1977).Fusing human rod outer segments from an eye enucleatedfor choroidal melanoma. Investigative Ophthalmology, 16,678-683.

Dowling, J. E., and Sidman, R. L. (1962). Inherited retinal

dystrophy in the rat. Journal of Cell Biology, 14, 73-109.Fain, G. L., Gold, G. H., and Dowling, J. E. (1976). Receptor

coupling in the toad retina. Cold Spring Harbour Symposiaon Quantitative Biology, 40, 547-561.

Falk, G., and Fatt, P. (1973). Changes in structure of thedisks of retinal rods in hypotonic solutions. Journal ofCell Science, 13, 787-799.

Gass, J. D. M. (1973). Drusen and disciform degeneration,macular detachment and degeneration. Archives ofOphthalmology, 90, 206-217.

Hayes, K. C. (1974). Retinal degeneration in monkeysinduced by deficiencies of vitamin E or A. InvestigativeOphthalmology, 13, 499-510.

Hogan, M. J. (1972). Role of the retinal pigment epitheliumin macular disease. Transactions of the American Academyof Ophthalmology and Otolaryngology, 76, 64-80.

Hogan, M. J., Alvarado, J. A., and Weddell, J. E. (1971).Histology of the Human Eye: An Atlas and Textbook.W. B. Saunders: Philadelphia.

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Ishikawa, T., and Yamada, E. (1970). The degradation ofthe photoreceptor outer segment within the pigmentepithelial cell of rat retina. Journal of Electron Microscopy,19, 85-91.

Kroll, A. J., and Machemer, R. (1968). Experimental retinaldetachment in the owl monkey: III. Electron microscopyof retina and pigment epithelium. American Journal ofOphthalmology, 66, 410-427.

Kuwabara, T. (1970). Retinal recovery from exposure tolight. American Journal of Ophthalmology, 70, 187-198.

Laties, A. M., and Enoch, J. M. (1977). An analysis of retinalreceptor orientation. I. Angular relationship of neighbour-ing photoreceptors. Investigative Ophthalmology, 10, 69-77.

Marshall, J. (1970a). Thermal and mechanical mechanismsin laser damage to the retina. Investigative Ophthalmology,9, 97-115.

Marshall, J. (1970b). Acid phosphatase activity in theretinal pigment epithelium. Vision Research, 10, 821-824.

Meek, G. (1963). In discussion of 'A scheme for sectionstaining in electron microscopy', by Mercer, E. M.Journal of the Royal Microscopical Society, 81, 184.

Pedler, C. M. H., and Tilly, R. (1967). The fine structure ofphotoreceptor discs. Vision Research, 7, 829-836.

Sarks, S. H. (1976). Ageing and degeneration in the macularregion: a clinico-pathological study. British Journal ofOphthalmology, 60, 324-341.

Spitznas, M., and Hogan, M. J. (1970). Outer segments ofphotoreceptors and the pigment epithelium: inter-relationship in the human eye. Archives of Ophthalmology,84, 810-819.

Steinberg, R. H., Wood, I., and Hogan, M. J. (1977).Pigment epithelial ensheathment and phagocytosis ofextra foveal cones in human retina. Philosophical Transac-tion of the Royal Society ofLondon, Series B, 277, 459-474.

Tormey, J. McD. (1964). Differences in membrane con-figuration between osmium tetroxide-fixed and glutaralde-hyde-fixed ciliary epithelium. Journal of Cell Biology, 23,658-664.

Young, R. W. (1971). Shedding of discs from outer segmentsin the rhesus monkey. Journal of Ultrastructure Research,34, 190-203.

Young, R. W. (1976). Visual cells and the concept of renewal.Investigative Ophthalmology, 15, 700-725.

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