A new perspective on Bruch's membrane and the retinal pigment epithelium

retinal pigment epithelium *

MICHAEL H. GOLDBAUM AND KENDALL MADDEN

Fromthe Division of Ophthalmologyat VeteransAdministration Medical Centerand University of California, San Diego, California, USA

SUMMARY Trypsin digestion of retinal pigment epithelium is a technique that bares Bruch's

membrane,

to allow topographical examination by scanning electron microscopy. Twenty-five humaneyes wereexamined. The zonula occludens of thepigment epitheliumwasclearlyseenasa surface feature, but attachment plaques at the sides and base were not visible. The adhesion between the pigment epithelium and the basal laminawasstrongerthan betweenthe basallamina and the restofBruch's membrane. Surface features of the basallamina, innercollagenouszone,

elastic layer, and outer collagenouszone were seen in away thatpreviously required anartist's representationconstructed from microscopic sections.

Since1844, when Karl Bruch describeda'structureless membrane' between the retinaandchoroid,' almost all anatomical examinations have been by light microscopy ortransmission electron microscopy.2- Most ofthese histological and electron microscopic sections were cross-sections or tangential sections through the membrane, whichwedescribetodayas having 5components:(1)basallamina of thepigment epithelium; (2) inner collagenous layer; (3) elastic layer; (4) outer collagenous layer; and (5) basal lamina of thechoriocapillaris,whereitis present.

Atechniquewas devised to loosen enzymatically pigment epithelium from Bruch'smembrane to allow evaluation of surface features byscanning electron microscopy, thereby offering a perspective viewof Bruch's membrane and its component layers. This technique also demonstrates surface features of pigmentepithelium that havenotbeendemonstrated previously. Scanning electron microscopyallowscon- ceptualisation of3-dimensional structure in a way that sections cannot. In thisreport the surface features ofBruch'smembrane and parts of the retinal pigment aredescribed.Inseparate reports the same techniques willbe used toevaluate regionalvariations, drusen, andaging changesinBruch'smembrane.

Correspondence to Michael H. Goldbaum, MD, 225 Dickinson Street(H-898) San Diego,California 92103, USA.

*Presented ^{in part atthe}Association for Research in Vision and Ophthalmology.Sarasota, Florida,4 May 1979.

Methods andmaterials

Twenty-five eyes were obtained from the eye bank andplaced in chilled bufferedglutaraldehyde 1% and paraformaldehyde 1% (buffered to pH 7A45 with 0-1 M monobasic and dibasic phosphate) between 50 minutes and 3 hours25minutes afterdeath. The corneas ofmost of the eyes were removed prior to fixation. The donorsrangedinagefrom15 to 83 years.

After fixation the eyes were sectioned coronally through theequator.Eachcalotte wasexamined with the stereoscopic microscope for drusen, pigment changes, and other abnormalities. Three specimens wereobtainedwithtrephines. A specimen containing thedisc and macular regionwasobtained with an 8 5 mm trephine. A specimen at the equator and a specimenstraddlingtheoraeachwereobtained with a6mmtrephine.

Tohelp localisation of the regionunderthe fovea thepiececontaining discandmaculawasnotchedat the edge superior and inferior to the fovea. The sensory retina was removed, and each piece was photographed.

Aftertheretinawasremoved each trephined piece was digested in buffered bovine pancreas trypsin 0-5% (Sigma) for 40 to 60 minutes at 37°C. The trypsin was buffered to pH 7 25 with 0-1 M Tris maleatebuffer in 0-02%ethylene diamine tetra-acetic acid. The retinal pigment epithelium was washed offBruch's membrane with a stream of phosphate 17

Fig.1 Scanningelectron g

microscopyofpigmentepithelium showingsmooth side walls and microvillioninnersurface.

Pockets inmicrovilli surrounded

rods. Microvilliatborderof.....JN

pockets(arrow)mattogetherto

formridgesthatsurroundtipof `77

rod.D=druse. (x1745).

bufferfrom apipette

(0-1

Mmonobasic and dibasic phosphateatpH7-45).Thespecimenwasthenpost- fixed in osmium tetroxide 2%.

Forscanningelectronmicroscopy aspecimenwas

dried inacritical-point bomb, coatedwith

gold

and palladium,and examinedby

mapping

at

progressively

increasing magnification. Fortransmission electron microscopy only, a specimen was dehydrated in increasing concentrations of ethanol, mounted in Epon812,thin-sectioned,and stainedwithleadcitrate anduranylacetate. Somespecimenswereexamined by correlated scanning and transmission electron

microscopy of thesamepiece.Afteraspecimenwas

examined by scanning microscopy it was placed in ethanol 100%, then mounted in Epon 812 for thin sectioning and staining.'° Other pieces were cutin half for separate processing for scanning electron microscopy and transmission electronmicroscopy.

Results

PIGMENT EPITHELIUM

The digestionprocess loosenedmostof the pigment epithelium from Bruch's membrane; irrigationwith

*. MV..:

Fig. 2 Transmissionelectron microscopyofpigmentepithelium and Bruch's membrane.

MV=microvilli, RPE=pigment epithelium,BI=basal infolding, BLR=basal laminaofpigment epithelium, ICZ=inner collagenouszone,EL=elastic lamina, OCZ=outer collagenous zone,BLE=basal laminaof endothelium ofchoriocapillaris.

(x8290). ^....

BLR

EL

* ^i> iP :' .e§ .roi...z

Fig. 3 Rodsinserting into plate ofmaterialonapical surface ofretinalpigment epithelium(arrow). ROS=rod

outer segment.(Magnification=x5965).

the

specimens

theretinacompletely separated from the

pigment epithelium, leaving pockets

where the outerreceptorsof the rodshad been. Themicrovilli

were

long

andhair-like between thesepockets."At the border of the

pockets

the villous processes

appeared

matted

together

ortook theformofa

ridge partially

wrapped around the pocket. In some

specimens

thevillous processeswerecoveredwitha

material,

presumably the

mucopolysaccharides

betweenouterreceptorsandthepigment

epithelium.

Thismaterial

appeared

as a

plate

with spacesfor the receptors

(Fig. 3).

The site ofthe zonulaoccludenswas visibleas a

band about500nmwideencirclingeachpigment cell

atthe

junction

betweentheapexandthesides

(Fig.

4).

Inmost

specimens

thisbandwas aflat

outpouching

ofthe cellmembraneatthe sides of thepigmentcell.

In otherspecimensthisbandappearedtoshrink from

processing

more than the sides of the cell, which sometimes

bulged

abovetheband.

The zonulaadherensandthe maculaeadherentes werenotvisibleonthesidesofthepigment

epithelium by

scanning electron microscopy. The smooth cell membrane on the sides of the pigment

epithelium bulged

overthemelaningranules(Fig.

1).

The basal surface of the pigment epitheliumwas

generally

not visible from the outside. In one

specimen a sheet ofremaining pigment

epithelium

was detached from the basal lamina and curled

Fig.4 Pigment epitheliumwitha bandatsiteofzonulaoccludens (ZO)surroundingeach cell at junctionbetween sides andapex.

The sides showsomedeterioration

fromexposuretotrypsin.(x3117).

t r

Fig. 5 Sheetofpigmentepitheliumthat haspeeledupfrom basal lamina.Anamorphous materialcoatsoutersurface of pigmentepithelium (OSR).Somebasalinfoldingisvisible (arrow). (x 2147).

Fig.7 Remnantsofbasal infoldingsattachedbyattachment plaques(arrow)topigment epithelium. (x 10286).

Fig. 6 Partiallydigestedpigmentepithelium.Remnant basalinfoldings(B I)onthebasallamina show that adhesion betweenbasallaminaand basalinfoldingwasstronger than integrityofcellinthis eye.(x5965).

Fig. 8 Basal laminapeelingawayfrominnercollagenous zone. At low power basal lamina appears likeaplastic membrane. (x 2684).

Fig. 9 Basal lamina at high magnification appearsgranular orvelvety. (x14316).

Fig. 10 Detachmentofpigment epithelium(RPE)exposing outer surfaceofbasallamina(BL)much in the mannerofaclinicalretinal pigmentepithelialdetachment.

Noteamorphousmaterial between pigmentepitheliumandbasal lamina(arrow).Noattachment sitesarevisible between basal lamina and inner collagenous zone.

Inthis specimen the retina remains, exposing outer segment(OS).

(x 1496).

Fig. 11 Where basallaminais

absent,

the innercollagenous

zoneisvisible,revealing tight interweaving ofcollagenfibres.

Thefibresoccasionallyappearjoinedtogetherlengthwise (arrow). (x 5368).

inwards. An amorphous material adhered to the externalsurface,leavingclefts wherebasalinfolding waspresent(Fig.5).

PIGMENT EPITHELIUM ADHESIONS TO BRUCH'S MEMBRANE

In many specimens the adhesion of the basal cell membrane tothe basallamina ofBruch's membrane was stronger than the integrity of the cell, leaving remnant basal cell membrane on the basallamina.

The internal aspect of this remnant membrane showedbasalinfolding(Fig.6). Transmission electron microscopy of the same piece showed attachment plaquesofthe flat parts of the cell basalmembraneto the basallamina(Fig. 7).

Broad expanses of the basal lamina were com- pletely bared following enzymatic digestion and irrigation. Theinner surfaceof thebasal laminawas smooth andshowed no evidence of the location of attachment plaques ofpigment epithelium (Figs. 8 and9).

BRUCH'S MEMBRANE

Four of the 5 layers of Bruch's membrane were

O.pm

Fig. 12 Withhigh magnification thefibres ofthe inner collagenouszoneappeartohaveanexternalrepresentation ofbanding. (x29825).

observed by scanning electron microscopy. The processofenzymaticdigestionandirrigation

exposed

mostof Bruch's membrane. 25 to50% ofthe basal laminawas lost from each specimen, allowingeasy visualisationoftheinnercollagenouszone.Therest ofBruch's membranegenerallyremainedintact. The shrinkage produced by the critical-point

processing

caused the edges of some specimens of Bruch's membranetocurlinwards, allowingvisualisationof theelasticlayerand theoutercollagenouslayer. The basal laminaofthechoriocapillarisendotheliumwas not seen.

Basal laminaofthepigment epithelium. By trans- mission electronmicroscopythebasal lamina showed

as astraight, finely-granularmembrane 40to100nm

thick(Fig. 1).Its appearancebytransmission electron microscopydidnotprepareonefor its appearance

by

scanningelectronmicroscopy.Withmoderatemagni- ficationthe basallaminaappearedlikeasmooththin plasticmembrane(Fig. 8).Athighmagnification,the membrane tookon amoregranularappearance

(Fig.

9).Both surfaces were smooth. There was no visible evidenceontheinnersurfacewherepigment

epithelial

attachmentswerelocated.Therewas noevidenceof

Fig. 13 Elasticlaminawith somecollagenfibres lyingon Fig. 14 CurlingupofpartofBruch'smembrane has innersurface. Someofinnercollagenfibresareinserted into exposedoutercollagenouslayer. Thecollagenfibresareless elastic layer and some join outercollagenouslayerby passing tightlywoven. (x5276).

throughholes inelastic lamina. (x8350).

insertion ofcollagen fibrils from theinner collagenous layerinto the basallamina(Fig. 10). The basal lamina tendedtoseparateeasily from the innercollagenous layer;theattachment between the basal lamina and the innercollagenous layer appearedtobe muchless firm than between the pigment epithelium and the basallamina(Figs.6,10).

Innercollagenous layer. At lowmagnification the innercollagenous layerappearedlikevelvet(Fig.8).

Atmoderatemagnification the collagen fibres in the innercollagenous layerformed atightlyinterwoven membrane (Fig. 11).The groundsubstancebetween thefibreswasnotvisible. Athigh magnification the collagen fibres appearedtohaveauniformthickness

ofabout70 nm(Fig. 12).There weretransverse ridges with a periodicity of about 60 nm, perhaps repre- sentingasurfacemanifestationof thebanding seenin these fibresby transmissionelectronmicroscopy.

Elastic layer. The elastic layer appeared to be composed ofcoarseandfinefibresmattedtogetherby some amorphous substance (Fig. 13). This layer formed an almost solid sheet with clefts and small holes. Collagen fibres from the inner collagenous

layer

appearedtobeinserted into the elasticlayerand alsotopassthroughholes in the elastic lamina to

join

withcollagenfibres in theoutercollagenous

layers.

Outercollagenous layer.Thecollagenfibres in the outer collagenous layer were less dense, finer in appearance,andmorelooselyinterwoven than those in theinnercollagenous layer (Fig. 14).Thecollagen network between the choriocapillaries appeared similar totheoutercollagenous layer,with which it

wascontinuous

(Fig. 15).

Discussion

The enzymatic looseningof the pigment epithelium allowed scanning electron microscopy of Bruch's membrane. Some of thepigment epitheliumdidnot washaway, andtheremainingcellsshowedvariable amountsofenzymeeffect, rangingfrom notvisibleto

severelydigested.

Themicrovilli around receptor elements have pre- viously been observed by transmission electron microscopytomattogether and form ramparts.9 The ridgewesawwithscanningelectronmicroscopy may

'Y"W:V.i..`--., _ON".

y-yR

N.R

...

i.MM PR

W.

Fig. 15 Outer collagenous layer is continuous with collagen

fibresbetween vessels in choroid. SplitinBruch's membrane has exposed choriocapillaris and collagen fibres between.

(x3517).

bethesamefeature. Onthe otherhandthere could be an enzyme artefact. Likewise the plate we sometimesobserved betweenpigmentepitheliumand rods and cones could be mucopolysaccharides or could be an enzyme artefact. The zonulaoccludens was asurfacefeature visibleas aband roundthe sides of the cell nearthe apex.213 We believe the zonula occludens has not previously been described as a visible surface feature of the retinal pigment epi- thelium.Itfunctionsas an outerblood-retinabarrier to large molecules.14-6 The sides of the pigment epithelium covered the pigment granules and were otherwise smooth; attachment plaques at the sides Fig. 16 Artist's representationofpigment epithelium and Bruch's membrane. Microvilli extend from apical surface ofpigmentepithelium, occasionally forming pockets forrod outersegment. Thezonula occludens barrier surrounds each cell,separating the sidemembrane and the apical surface.

The basal lamina of the pigment epitheliumis smoothlike a thinplasticmembrane. Basalinfoldings adheretobasal lamina, whichis nottightly attachedtotheinnercollagenous zone. Innercollagenousfibres are more tightly woven than outer

collagenous.fibres.

Elasticlamina forms a plate with holes between the2collagenous zones.

junction is also the site ofseparation for the clinical condition of retinal pigmentepithelial detachment.

Attachmentplaquesatthe basallayerof the pigment epithelium were seen in transmission electron microscopy, but were notvisible assurfacefeatures by scanning electron microscopy. The amorphous material observed on the outer surface of the cell basal membrane (Fig. 10) may be involved in the adhesion to the basal lamina. Therewasno visible mechancal attachment between the smooth outer surface 6fthebasal lamina andthe rest ofBruch's membrane(Fig. 10).

The tightly woven collagen fibres of the inner collagenous layerandthe denseelasticlayer by their appearance canbe surmisedto act as a flexible yet stable platform in2dimensionstosupporttheretina, muchasthe plasticfilm base acts as asupportfor the emulsion inphotographic film. Another functionthat can be hypothesised is that these layers act as a mechnical barrier that keeps the choriocapillaries away from the retina. Fig. 16 representsan artist's concept of the composite findings of scanning and transmissionelectronmicroscopy.

Dale Deg assistedindevising techniques to expose the layers in Bruch's membrane.

This work was supported in part by grant NIH EY 02434 and VA grant MRIS 3192.

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epithelium.

M H Goldbaum and K Madden

doi: 10.1136/bjo.66.1.17

1982 66: 17-25 Br J Ophthalmol

http://bjo.bmj.com/content/66/1/17

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