TypeIV inBruch's membrane - British Journal of …bjo.bmj.com/content/bjophthalmol/76/10/607.full.pdf · from freshly enucleated eyes of five uveal melanoma patients, a patient with

British journal ofOphthalmology 1992; 76: 607-614

Type IV collagen and laminin in Bruch's membraneand basal linear deposit in the human macula

G E Marshall, A G P Konstas, G G Reid, J G Edwards,W R Lee

AbstractTissue obtained from the macula in 10 humaneyes (53-77 years) was used for an investigationinto the extracellular matrices of the retinalpigment epithelium (RPE), Bruch's membrane,and the choriocapillaris. The ultrastructuraldistribution of type IV collagen and lamininwas documented using immunogold labelling.Labelling for type IV collagen was stronglypositive in all the specimens in the basementmembranes of the choriocapillaris but not thatof the RPE where labelling was either weak orabsent. Laminin was localised to deposits ofgranular material in Bruch's membrane butwas absent from the basement membrane ofthe RPE and the choriocapillaris. Basal lineardeposit, observed in three cases, demonstratedlabelling for laminin but not for type IV

collagen. The series was too small forcorrelation of these morphological changeswith age.

(BrJ7 Ophthalmol 1992; 76: 607-614)

Departments ofOphthalmology, CellBiology and Pathology,University ofGlasgow,Glasgow Gil 6NTG E MarshallA G P KonstasG G ReidJ G EdwardsW R LeeCorrespondence to:Dr G E Marshall, Departmentof Ophthalmology, Universityof Glasgow, Glasgow GI6NT.

Accepted for publication23 April 1992

Vision can be affected in the elderly by a varietyof pathological changes which interfere with thefunction ofthe retinal pigment epithelium (RPE)and the maintenance of the photoreceptors.Photoreceptor atrophy occurs when an abnormalextracellular matrix - for example, drusen,accumulates between the pigment epitheliumand Bruch's membrane. Drusen are clinicallywell recognised and can be classified as hard,soft, calcified, and diffuse according to histo-logical criteria. ' However, a morphologicaldistinction can be made between drusen andbasal linear deposit. Various authors24 have usedthe term 'basal linear deposit' for an accumu-lation with specific appearances (after PAS andMallory trichrome stains) beneath the RPE. Theprecise biochemical composition of the basallinear deposit is not known and identification ofthe constituents is essential to an understandingof the pathogenesis of age-related maculardegeneration.

Studies using transmission electron and lightmicroscopy have adequately documented themorphology of the age-related changes inBruch's membrane.258 The initial change is an

accumulation of vesicles and granular andfilamentous material in the inner collagenouszone. This accumulation progressively increaseswith age until it occupies both collagenous zones.

Various attempts have been made to determinethe biochemical nature ofthe age-related depositsin Bruch's membrane outside the macularregion9-'3 but it is not known iftheir accumulationis the cause or the result of RPE dysfunction.Since Bruch's membrane may act as a selectivebarrier to the movement of molecules from the

choroidal circulation to the outer retina, 14alterations in its composition could haveimportant physiological consequences.

Age-related thickening and degeneration inBruch's membrane,7'15 drusen formation,'6 andbasal linear deposits3 each appear to provide amatrix which stimulates ingrowth of macro-phages and endothelial cells. Vascular ingrowthis an important complication of age-relatedmacular degeneration and is a major cause ofblindness in the elderly population ofthe Westernworld.7 18 Bruch's membrane acts as a barrier tosubretinal neovascularisation and breaks in thisstructure significantly increase the incidence ofgrowth of new vessels.7 The differences betweenthe factors causing senile degenerative atrophyand disciform degeneration are poorly under-stood.4 It is now accepted that type IV collagenand laminin are matrix constituents whichfacilitate endothelial cell movement in vitro.'922Thus knowledge of the precise biochemicalcomposition of the age-related deposits inBruch's membrane will allow an evaluation oftheir potential influence on vascular ingrowth indisciform degeneration. As early forms of basallinear deposit have ultrastructural similarities tobasement membrane2 it was considered likelythat the deposit would contain type IV collagenand laminin which are the principal componentsof many basement membranes. In this study weinvestigated the fine, structural distribution ofcollagen type IV and laminin in Bruch's mem-brane of aged human macula.

Materials and methodsNormal aged macular tissue was obtained bothfrom freshly enucleated eyes of five uvealmelanoma patients, a patient with a cornealulcer, and from four postmortem eyes. The agerange ofthe subjects was between 53 and 77 years(Table 1). Routine macroscopic and microscopicexamination excluded the presence of anysecondary disease process which could haveaffected macular morphology.

Surgically enucleated eyes were fixed

Table I Clinical details ofsurgical enucleations andpostmortem eyes

Case no Age Sex Indication for enucleation

1 53 F CBM2 54 F CBM3 58 M PM4 59 M PM5 60 M PM6 64 M PM BLD7 71 F PM BLD8 73 M CM BLD9 75 F CBM10 77 F CM

CBM=ciliary body melanoma; CM=choroidal melanoma;PM=postmortem; BLD= basal linear deposit.

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Marshall, Konstas, Reid, Edwards, Lee

immediately after enucleation in freshly preparedfixative of 4% paraformaldehyde (PFA) plus0-2-1 0% glutaraldehyde in 0-1 M Sorensen'sphosphate buffer or cacodylate buffer (pH 7 4)with 5% sucrose added; details of fixation are

provided elsewhere.23 The postmortem eyes

were obtained from the Greek Eye Bank(University Department of Ophthalmology,

Figure I The paramaculararea in a 53-year-oldfemalewith a ciliary bodymelanoma. The choroidalvessels are thin-walled andBruch's membrane is notthickened (toluidine blue,x24).

Thessaloniki) and fixed within 1 5 to 9 hours ofdeath following removal of the cornea fortransplantation. Fixation was achieved with0 5% glutaraldehyde plus 4% formalin inphosphate buffer pH 7-4 at room temperaturefor 2 hours. Details of the medical and ocularhistory of the postmortem cases were obtainedfrom close relatives and subsequent macroscopicexamination revealed a normal macula and disc.The macula region was dissected from each

eye and in the majority of cases the retinaremained attached. Radial blocks were taken andprocessed for London resin white (LR white)embedding and cryoultramicrotomy.23 LR whiteis a hydrophilic resin that is used for immuno-cytochemical studies owing to its property ofpreserving tissue antigenicity.24

IMMUNOCYTOCHEMISTRYPolyvalent goat antibodies against type IVcollagen were raised against human and bovinetype IV collagen and supplied by SouthernBiotechnologies (Bionuclear Services Ltd). Theantibody was affinity purified and cross absorbedagainst types I, II, III, and V collagen whichwere purified from human tissue. The specificityof this antibody to human type IV collagen hasbeen confirmed by the supplier using indirectenzyme linked immunosorbent assay (ELISA).

Polyvalent rabbit antibodies against lamininwere supplied by Heyl (Germany) and had beenraised in rabbits by multiple injections ofhumanlaminin. The serum has been tested by thesupplier with the following immunologicalassays: ELISA, immunoblot, and immuno-histology (fluorescence, APAAP). The antibodydilutions in Tris buffer plus 1% BSA, determinedby previous experimental work, were between1:40 and 1:150 for type IV collagen and lamininantibodies at dilutions between 1:30 and 1:100.These antibodies have been successfullyemployed in our laboratory for immunogoldelectron microscopic studies of various oculartissues.25-28 The precise immunocytochemicalprocedure has been described by Marshall et al. 27

CONTROLSNegative controls comprised substitution of theprimary antiserum with normal goat and rabbitserum (Sigma) at the same dilutions. Retinalvessels were used as an internal positive control(within the same tissue section) for type IVcollagen which is located in the basementmembranes within the vessel wall." The base-ment membranes of the ciliary epithelium wereused as an external positive control for type IVcollagen and laminin.2829 We consider internalpositive controls to be even more reliable thanexternal positive controls from another tissueblock.Our criteria for positive localisation required

the restriction of immunogold particles todiscrete structures. Labelling was considered tobe non-specific if a significant number ofimmunogold particles were present on internalnegative controls. Internal negative controlscomprised cell nuclei, mitochondria, pigmentgranules, and red blood cells: on the basis of

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Type IVcollagen and laminin in Bruch's membrane and basal linear deposit in the human macula

M,;tit,, x IM. < k-9i j$; previous investigations these had been found tobe the first structures to exhibit non-specificlabelling.

.~~~~~~ 7,Results-M> - The use of different concentrations of

CvL glutaraldehyde in surgically enucleated eyes didnot alter the pattern or intensity of immunogold

D tlabelling for type IV collagen and laminin. TheSllNr,X Sw i quality of preservation of cells in postmortem

E ~~~~~~~~~specimens was poorer than that in surgicallyenucleated eyes, but in all the specimens the

*; Si; structure was morphologically satisfactory by' ~~light microscopy (Fig 1). The addition of 5%

4 > l ightsucrose to the cacodylate buffer eliminated thepreviously documented superiority of phosphate

r _ * w C Sbuffer in ultrastructural preservation .mn- ~~~~~There was no apparent difference in immuno-

labelling of collagen type IV and laminin whencomparing sections from LR white processedtissue with ultrathin sections of frozen tissue. LRwhite embedding was predominantly used

4 ii !because it was much less technically demandingin terms of preservation of ultrastructure in the

material used in the present study. Our previous,- studies in aged ocular tissues have demonstrated_____ ~~~~~~~~~~~~~~similarresults with both techniques for laminin

and type IV collagen. Therefore for the study of

Figure 2 Type IV collagen distribution in Bruch's membrane. Immunogold particle density embedding was considered the technique ofover the RPE basement membrane (bm) is considerably less than that over the basementmembrane (BM) of the choriocapillaris. A small amount oftype IV collagen is also present in choice. The employment of both techniquesthe inner collagenous layer (ICL), the elastic layer (E), and the outer collagenous layer (OCL), served to consolidate the results. However, thethat of the elastic layer occurring in a space between elastic fibrils. Note association offibrous illustrations for the present communication werebanded material with elastic layer as well as its presence in the outer collagenous layer (arrows). prepared from LR white embedded material.Coated vesicle-like bodies can be seen in both the inner and outer collagenous layers(arrowheads). Coated membrane fragments (F) are restricted to the outer collagenous layer(bar=I Fm).

'0111%~..0L...Figure 3 Fine structural T'localisation oflaminin in.......Bruch's membrane. b;mImmunogold particles are 0

absentfrom the basementmembranes oftheRPE (bm)and the choriocapillaris(BM). Some degree of _labelling is present in the A_inner collagenous layer ~(ICL) particularly at itsinterfacewith theRPE I Lbasement membrane(arrowheads). Note theabsence ofimmunogoldparticles from the elasticlayer (E). Labelling of theouter collagenous layer(OCL) is concentratedmainly to granular depositsin the region of tsnterface :; ;with the choriocapillarybasement membrane (BM).Coated vesicle-like bodies E.(V) and coated membranebound bodies (M) are free oflabel (bar= 1 ptm.) Inset A:Laminin labelling at the ...

interface between the RPE `0: W.basement membrane (bm)andtheinnercollaaenous~~~~~~~~~~~~~~~~~~~~~~A

small electron dense plaques ai

Immunogold particlestocalising laminin are largelyrestricted to the periphery of rik

fibrous banded material ....f..-(bar=0O5 [sm).

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Marshall, Konstas, Reid, Edwards, Lee610

NOMENCLATUREIn the description of type IV collagen andlaminin distribution in Bruch's membrane wehave adopted the nomenclature proposed byKillingsworth5 to classify the various depositsassociated with aging of Bruch's membrane.According to this classification 'coated vesicle-like bodies' are circular bodies composed of asingle membrane lining an electronlucent coreand occurring in two sizes (70 nm and 110 nm).'Coated membrane-bound bodies' (400 to 2500nm diameter) contain fine granular material,droplets, and coated vesicle-like bodies. 'Coatedmembrane fragments' are thought to be theremnants of ruptured coated membrane-boundbodies.5

IMMUNOCYTOCHEMISTRY

Basement membrane ofthe RPEIn some specimens the basement membrane ofthe RPE exhibited weak labelling with type IVcollagen antibodies (Fig 2) but this finding wasnot uniform in all the cases studied as labellingwas absent from other specimens. Such variationin labelling could not be related to the source oftissue (that is, surgical enucleations or post-mortem specimens) nor to the concentration offixative employed. The most surprising findingwas that laminin was not identified in the RPEbasement membrane in any of the specimensinvestigated (Figs 3-6).

Bruch's membraneA small amount of labelling for type IV collagenwas present in the inner collagenous layer, theelastic layer, and the outer collagenous layer ofBruch's membrane. This was a consistent featurein all the specimens studied. Although labellingwas present in the elastic layer, immunogoldparticles were not located to the elastic fibres butrather to the matrix surrounding these fibres(Fig 2). Immunogold labelling for type IVcollagen was absent from coated vesicle-likebodies, coated membrane bound bodies, coatedmembrane fragments, and fibrous bandedmaterial. Laminin was localised to deposits offine granular-like material present in the innercollagenous layer. More frequently laminin wasdispersed within the fine granular-like materialbetween the outer collagenous layer and thebasement membrane of the choriocapillaris (Fig3). Small discrete patches of laminin labellingwere also observed in the inner collagenous layerimmediately adjacent to the RPE basementmembrane (Fig 3, inset A). These patches oflabelling were spaced in a manner which wasalmost regular. The only difference in thestructure of these patches and the innercollagenous layer was a small augmentation inelectron density.

Laminin was also associated with theperiphery of fibrous banded material presentwithin Bruch's membrane, labelling beinggenerally absent from the actual striations of thismaterial (Fig 3, inset B). We considered thatfibrous banded material was quite distinct fromlong-spacing collagen in its location, structure,

Figure 4 Laminin distribution in major blood vessel in thechoroid. Laminin localised to discrete clumps ofgranular-likematerial (arrowed) between the three layers ofmyocytes (M)within the vessel wall. Note absence oflabelfrom red bloodcell (RBC). (end=endothelium ofblood vessel (bar=I [tm).

and periodicity. Long-spacing collagen waspresent between the outer collagenous layer andthe basement membrane of the choriocapillaris.Fibrous banded material was located within boththe inner and outer collagenous layers and some-times in association with the elastic layer (Fig 2).

In contrast with long-spacing collagen, fibrousbanded material had a significantly shorterperiodicity and lacked the fine structure of thelatter. No association of laminin was seen withlong spacing collagen within Bruch's membrane.Similarly, laminin was absent from the elasticfibres in the elastic layer (Fig 3).

ChoriocapillarisThere was considerable degree of variation in thethickness of the basement membrane of theendothelial cells and this could not always beattributed to the plane of section of the chorio-capillaris. Type IV collagen was clearly localisedto the basement membranes around the circum-ference of the choriocapillaris and labelling waspresent throughout the entire width of thebasement membrane. In contrast to the RPEbasement membrane, the basement membraneof the choriocapillaris exhibited intense labellingwith type IV collagen antibodies (Fig 2). Anti-body labelling for laminin was so sparse as to beregarded as negative.

Choroidal stromaType IV collagen and laminin were localised tothe basement membranes of the large choroidalvessels. Type IV collagen was also localised to allbasement membranes present in the stroma,some of which were not associated with cells but

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Figure 5 Immunogold J.VA< .localisation oflaminin in .*earlyform ofbasal lineardeposit. LabellingisFrestricted to thecircumference of thisamorphous basementmembrane-like materialwhich has notyetseparated the retinalpigment epithelium (RPE)from its basementmembrane (bar= I [tm). ____

Figure 6 Laminin Kpresent within filamentousforn ofimmature basallinear deposit (arrowed)(bar= I ptm).

Figure 7 Early stage basallinear deposit exhibitinggranular-like morphology islabelled throughout withantilaminin antibodies. Notenumerous basal infoldingsseparating depositfrom........basement membrane (bmi) of :retinal pigment epithelium(RPE). Note absence oflamininfromRPE basementmembrane (bar=I m). h. .. ,

were lying free amongst collagen fibrils of thestroma. Small isolated deposits of fine granularmaterial, similar in appearance to basal lineardeposit (see below), were occasionally seen in thestroma between large blood vessels and werepositive for laminin (Fig 4). Elastic fibres withinthe walls of large blood vessels were free ofimmunogold particles for laminin or type IVcollagen. Considerably larger deposits ofgranular material similar to those observed inBruch's membrane were frequently present inthe intercapillary zone. These deposits appearedto be associated with choroidal stroma ratherthan the outer collagenous layer.

Basal linear depositBasal linear deposit was observed in three out ofthe 10 specimens (cases 6-8, see Table 1). Smallsized basal linear deposit could be divided intothree types on the basis of their morphologicalappearance: basement membrane-like, granular-

like, and filamentous-like. These deposits werewithin basal infolds and were not accompaniedby a separation of the RPE from its basementmembrane. Two distinct labelling patterns ofthese deposits were then noted with lamininantibodies. Labelling for laminin was restrictedto the margins of the basement membrane-likebasal linear deposit (Fig 5) but was presentthroughout the entire matrix of the granular-like(Fig 6) and fine fibrillar-like structures (Fig 7).

Larger forms of basal linear deposit containeda mixture of basement membrane-like, finegranular-like, and fine fibrillar-like material.These deposits were invariably accompanied bya separation of the RPE from its basementmembrane (Fig 8). The labelling pattern of thelarge deposits for laminin was similar to that ofthe small deposits: the margins of basement-likematerial and the entire matrix of the finegranular-like and fine fibrillar-like material werelabelled (Fig 8). Laminin was absent fromprofiles of long-spacing collagen associated with

!L A-., ... e . 1 . S0b00

Figure 8 More advancedform ofbasal linear depositlabelled with lamininantibodies. Immunogoldparticles are overfilamentous/granularmaterial, but are absentfronamorphous basementmembrane-like material.Note absence oflabellingfrom RPE basementmembrane (bm) and long-spacing collagen (arrowed).The cell basement membraneis shown by arrowheads.ICL = inner collagenouslayer, RPE=retinalpigment epithelium(bar=lIsm).

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Figure 9 Normal rabbitserum control. Note lack ofimmunogold particles frombasal linear deposit (BLD)and cell nucleus (Nu). Somenon-specific labelling ispresent in Bruch's membrane(Br) (bar= I pm).

these large basal linear deposits (Fig 8). Immuno-gold labelling for type IV collagen was notassociated with any of the basal linear depositsdescribed above.

CONTROLSImmunogold labelling was sparse and non-

Figure 10 Externalpositive control for laminin.Immunogold particles aredistributed throughout thebasement membrane ofthepigmented ciliary epithelium(PE)(bar=1Im).

specific in both the normal goat serum andnormal rabbit serum negative controls (Fig 9).Non-specific labelling tended to be greater innormal rabbit serum controls than in normalgoat serum controls. Type IV collagen wasdemonstrated in the basement membranes of theretinal vessels (internal positive control). In thecidiary body control tissue, collagen type IV andlaminin were observed on the basementmembranes ofthe pigmented and non-pigmentedciliary epithelium (Fig 10). Laminin was alsolocalised to zonular fibres arising from the ciliarybody.

Discussion

MACULAPrevious studies have been conducted on thedistribution of collagen type IV and laminin inBruch's membrane of the human eye'>-" buthave not referred to the macular region. To thebest of our knowledge the present study is thefirst to concentrate on normal aged humanmacula. The importance of the macula in retinaldisease merits separate consideration and thisstudy is intended to provide baseline informationfor further investigation of age-related maculardegeneration. It cannot be assumed that thedistribution of extracellular matrix componentsin the macular choroid and RPE is identical tothat of the extramacular regions, particularlysince there are obvious differences in functionalanatomy.

CHOROIDThe overall distribution of type IV collagen seenin our aged macular tissue was similar to that ofDas et all" in their fine structural immunogoldstudy of extramacular human tissue. In theirstudy and the present study labelling was moreintense in the basement membrane of the chorio-capillaris when compared with that of the RPEbasement membrane. A similarity was alsoobserved in the small amount of labelling of thecollagenous layers.Das et al " also localised laminin to the

choriocapillary and RPE basement membranesbut labelling was 'far less intense than that seenwith type IV collagen'. In our study lamininlabelling of these two basement membranes inthe macular region was of insufficient intensityto be considered as positive. Laminin, a largemultidomain glycoprotein, is a major structuralbasement membrane component30 and numerousbiological functions of basement membranes arecontrolled by its presence.920 However,although collagen type IV, laminin, nidogen,and heparan sulphate proteoglycan are the bestknown components of basement membranes,the molecular architecture of basementmembranes is incompletely understood.30 Inaddition, a variation in composition has beendemonstrated in basement membranes fromdifferent sites.30 The localisation of laminin bothto discrete structures within Bruch's membraneand to basal linear deposit is unique to thepresent study.

Light microscopic immunohistochemistry of

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human choroid indicated the presence of bothtype IV collagen and laminin in Bruch'smembrane.'012 Newsome et al,'0 in an immuno-fluorescene study, stated that there was abilaminar staining pattern for these twocomponents. Unfortunately, the lack ofresolution with immunofluorescent labellingdoes not permit a distinction between theconstituent layers of Bruch's membrane. Thebilaminar immunofluorescent pattern can beexplained by an early deposition of basal lineardeposit on the basal aspect of the RPE cells andin granular deposits in close apposition with thechoriocapillaris as demonstrated by electronmicroscopy in the present study. There are alarge number of alternative explanations for thedifferences in results obtained by immunocyto-chemistry, such as loss of antigenicity due tofixation, relocation of antigen in unfixedmaterial, and differences in antibody specificity.

BASAL LINEAR DEPOSITVarious types of drusen and basal linear deposithave been recognised histopathologically butclinicopathological correlations have beensparse.' At present no satisfactory classificationexists and identification of the constituents ofthese deposits may elucidate their origin. Disci-form macular degeneration represents a responseof the posterior pole tissues to pre-existingdisorder of the subretinal structures. Forexample, one proposal is that the process leadingto disciform degeneration begins with thickeningof the inner aspect of Bruch's membrane owingto synthesis of abnormal basement membrane-like material. 'Though basal linear deposit,3 or alternatively,

diffuse soft drusen,3' has been recognised bylight microscopists for many years and the finestructure was first described in 1964 by Lerche,3the nature remains enigmatic. Sarks3"4 sug-gested that basal linear deposit represents aprogressive disturbance in the metabolism of theRPE and that it may exert an influence onvascular ingrowth in disciform degeneration.3 Inits earliest form basal linear deposit is similar inmorphology to basement membrane materialand it is acceptable to speculate that basal lineardeposit is a secretory product of RPE cells.2 Inmore advanced stages basal linear deposit appearsto be augmented by cellular deposit derivedmainly from RPE cell membranes2 and long-spacing collagen.33 The association of lamininwith two constituents of basal linear deposit wasclearly demonstrated in the present study.Though in this investigation early forms of basallinear deposit possessed a similar morphology tothat of basement membranes, type IV collagenwas absent from these deposits. Thus basal lineardeposit was biochemically distinct from the RPEbasement membrane since the latter structurepossessed a variable amount of type IV collagenbut not laminin.Laminin was a component of all forms of basal

linear deposit which was observed between theRPE cell membrane and its basement membrane.Since laminin is an extracellular product asdistinct from a cellular constituent, it can beconcluded that early forms of basal linear deposit

are quite distinct from a mere deposition of cellbreakdown products. It is also reasonable toassume that laminin is a disordered syntheticproduct ofRPE cells due to its close proximity tothe basal cytoplasmic membrane.

LAMININ IN BRUCH'S MEMBRANEThe occurrence of laminin in deposits in theinner collagenous layer and between the outercollagenous layer and the basement membrane ofthe choriocapillaris appears to be unique to theaged macula. As these deposits were considerablymore common in intercapillary spaces, they mayrepresent remnants of retreating chorio-capillaris. Alternatively the deposits may be theproduct of a senescent RPE. The latter theory issupported by embryonic studies which haveshown that in the development of Bruch'smembrane there is an extension outward onelayer at a time from the basement-membrane ofthe RPE.34 Since the RPE has been shown in cellculture studies to produce all of the extracellularmatrixcomponents present in Bruch'smembraneit is reasonable to assume that much, if not all, ofBruch's membrane is laid down by the RPE.'2 Itis therefore conceivable that laminin depositsobserved in our study within Bruch's membraneare the synthetic products of RPE cells and arenot derived by diffusion from the chorio-capillaris. It may be that such deposits arepeculiar to aging and in being secreted in amonomeric form have polymerized withinBruch's membrane. On no occasion was anasssociation observed between laminin and theelastic tissue either in Bruch's membrane or inthe supply vessels of the macular choroid.

In previous studies ofaged human ciliary bodyand outflow system we noted plaques of extra-cellular matrix material in the tips oflongitudinalciliary muscle fibres29 and in the cribriformlayer.26 These plaques bear a striking resemblanceto the early forms of basal linear deposit seen inthis study and in each location the extracellularmatrix deposits were morphologically similar.The immunolabelling patterns for laminin andtype IV collagen were also similar. It is interestingto speculate that since in all three regions thetissues act as a barrier to fluid drainage, meta-bolic byproducts may stimulate extracellularmatrix deposition.

Finally, it is appropriate to consider thesignificance of the present study in relation toadhesion of the RPE to Bruch's membrane. Theoccurrence of discrete patches of lamininlabelling between the inner collagenous layer andthe RPE basement membrane could be attributedto the presence of anchoring plaques in arudimentary basement membrane complex. Wehave observed a similar labelling pattern in thecorneal epithelial basement membranecomplex.27 That the RPE is firmly adherent to itsbasement membrane is highlighted in the clinicalcondition of RPE detachment: here the site ofcleavage occurs between the RPE basementmembrane and the inner collagenous layer ratherthan between the RPE and its basementmembrane.35 This preferential adherence hasbeen confirmed by scanning electron microscopestudies ofRPE exposed to trypsin digestion: the

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basement membrane separated from the innercollagenous layer rather than the RPE.3 Firmadherence of the RPE to its basement membraneis presumably mediated by hemidesmosomeswhich have been described in the basal cellprocesses of the RPE.36 This in turn wouldsuggest the existence of a basement membranecomplex with anchoring fibrils and anchoringplaques such as that seen in corneal epithe-lium.3738 In an immunoelectron microscope studyof aged human cornea we were able to demon-strate the presence of laminin in the anchoringplaques of the basement membrane complex.27We would therefore suggest that labellingobserved between the RPE basement complexand the inner collagenous zone may be due to thepresence of anchoring plaques.We are pleased to acknowledge the technical assistance of MrsSophia Cameron. The efficiency of Dr T Kardasopoulos inproviding the autopsy specimens is greatly appreciated. This workwas supported by the RNIB and the Sir Jules Thorn CharitableTrust.

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16 Gass JDM. Drusen and disciform macular detachment anddegeneration. Arch Ophthalmol 1973; 90: 206-17.

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18 Bird AC. Recent advances in the treatment of senile disciformmacular degeneration by photocoagulation. BrJ3 Ophthalmol1974; 58: 367-76.

19 Herbst TJ, McCarthy JB, Tsilibary EC, Furcht LT.Differential effects of laminin, intact type IV collagen, andspecific domains of type IV collagen on endothelial celladhesion and migration. J CellBiol 1988; 106: 1365-73.

20 Panayotou G, End P, Aumailley M, Timpl R, Engel J.Domains of laminin with growth-factor activity. Cell 1989;56: 93-101.

21 Kleinman HK, Sephel GC, Tashiro KI, Weeks BS, BurrowsBA, Adler SH, et al. Laminin in neuronal development. AnnNYAcad Sci 1990; 580: 302-10.

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