a perspective on bruch's membrane and the retinalpigment ... · pigment epithelium...

British Journal of Ophthalmology, 1982, 66, 17-25

A new perspective on Bruch's membrane and theretinal pigment epithelium *MICHAEL H. GOLDBAUM AND KENDALL MADDEN

From the Division of Ophthalmology at Veterans Administration Medical Center andUniversity of California, San Diego, California, USA

SUMMARY Trypsin digestion of retinal pigment epithelium is a technique that bares Bruch'smembrane, to allow topographical examination by scanning electron microscopy. Twenty-fivehuman eyes were examined. The zonula occludens of the pigment epithelium was clearly seen as asurface feature, but attachment plaques at the sides and base were not visible. The adhesionbetween the pigment epithelium and the basal lamina was stronger than between the basal laminaand the rest of Bruch's membrane. Surface features of the basal lamina, inner collagenous zone,elastic layer, and outer collagenous zone were seen in a way that previously required an artist'srepresentation constructed from microscopic sections.

Since 1844, when Karl Bruch described a 'structurelessmembrane' between the retina and choroid,' almostall anatomical examinations have been by lightmicroscopy or transmission electron microscopy.2-Most of these histological and electron microscopicsections were cross-sections or tangential sectionsthrough the membrane, which we describe today ashaving 5 components: (1) basal lamina of the pigmentepithelium; (2) inner collagenous layer; (3) elasticlayer; (4) outer collagenous layer; and (5) basallamina of the choriocapillaris, where it is present.A technique was devised to loosen enzymatically

pigment epithelium from Bruch's membrane to allowevaluation of surface features by scanning electronmicroscopy, thereby offering a perspective view ofBruch's membrane and its component layers. Thistechnique also demonstrates surface features ofpigment epithelium that have not been demonstratedpreviously. Scanning electron microscopy allows con-ceptualisation of 3-dimensional structure in a waythat sections cannot. In this report the surface featuresof Bruch's membrane and parts of the retinal pigmentare described. In separate reports the same techniqueswill be used to evaluate regional variations, drusen,and aging changes in Bruch's membrane.

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

*Presented in part at the Association for Research in Vision andOphthalmology. Sarasota, Florida, 4 May 1979.

Methods and materials

Twenty-five eyes were obtained from the eye bankand placed in chilled buffered glutaraldehyde 1% andparaformaldehyde 1% (buffered to pH 7A45 with0-1 M monobasic and dibasic phosphate) between50 minutes and 3 hours 25 minutes after death. Thecorneas of most of the eyes were removed prior tofixation. The donors ranged in age from 15 to 83 years.

After fixation the eyes were sectioned coronallythrough the equator. Each calotte was examined withthe stereoscopic microscope for drusen, pigmentchanges, and other abnormalities. Three specimenswere obtained with trephines. A specimen containingthe disc and macular region was obtained with an 8 5mm trephine. A specimen at the equator and aspecimen straddling the ora each were obtained witha 6 mm trephine.To help localisation of the region under the fovea

the piece containing disc and macula was notched atthe edge superior and inferior to the fovea. Thesensory retina was removed, and each piece wasphotographed.

After the retina was removed each trephined piecewas digested in buffered bovine pancreas trypsin0-5% (Sigma) for 40 to 60 minutes at 37°C. Thetrypsin was buffered to pH 7 25 with 0-1 M Trismaleate buffer in 0-02% ethylene diamine tetra-aceticacid. The retinal pigment epithelium was washedoff Bruch's membrane with a stream of phosphate

17

on June 16, 2020 by guest. Protected by copyright.

http://bjo.bmj.com

/B

r J Ophthalm

ol: first published as 10.1136/bjo.66.1.17 on 1 January 1982. Dow

nloaded from

http://bjo.bmj.com/

18 Michael H. Goldbaum and Kendall Madden

Fig. 1 Scanning electron gmicroscopy ofpigment epitheliumshowing smooth side walls andmicrovilli on inner surface.Pockets in microvilli surroundedrods. Microvilli at borderof....... ..JN

pockets (arrow) mat together toform ridges that surround tip of `77rod. D=druse. (x 1745).

buffer from a pipette (0-1 M monobasic and dibasicphosphate at pH 7-45). The specimen was then post-fixed in osmium tetroxide 2%.For scanning electron microscopy a specimen was

dried in a critical-point bomb, coated with gold andpalladium, and examined by mapping at progressivelyincreasing magnification. For transmission electronmicroscopy only, a specimen was dehydrated inincreasing concentrations of ethanol, mounted inEpon 812, thin-sectioned, and stainedwith lead citrateand uranyl acetate. Some specimens were examinedby correlated scanning and transmission electron

microscopy of the same piece. After a specimen wasexamined by scanning microscopy it was placed inethanol 100%, then mounted in Epon 812 for thinsectioning and staining.'° Other pieces were cut inhalf for separate processing for scanning electronmicroscopy and transmission electron microscopy.

Results

PIGMENT EPITHELIUMThe digestion process loosened most of the pigmentepithelium from Bruch's membrane; irrigation with

*.MV..:

Fig. 2 Transmission electronmicroscopy ofpigment epitheliumand Bruch's membrane.MV=microvilli, RPE=pigmentepithelium, BI=basal infolding,BLR=basal lamina ofpigmentepithelium, ICZ=innercollagenous zone, EL= elasticlamina, OCZ=outer collagenouszone, BLE=basal lamina ofendothelium ofchoriocapillaris.(x 8290). ....

BLR

EL

* ^i> iP :' .e§ .roi...z


http://bjo.bmj.com

/B

r J Ophthalm


nloaded from

http://bjo.bmj.com/

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

Fig. 3 Rods inserting into plate ofmaterial on apicalsurface ofretinalpigment epithelium (arrow). ROS=rodouter segment. (Magnification= x 5965).

phosphate buffer exposed broad expanses of Bruch'smembrane. Patches of pigment epithelium remainedattached to Bruch's membrane.

Microvillous processes extended from the apex ofthe pigment epithelial cells (Figs. 1 and 2). In most ofthe specimens the retina completely separated fromthe pigment epithelium, leaving pockets where theouter receptors of the rods had been. The microvilliwere long and hair-like between these pockets." Atthe border of the pockets the villous processesappeared matted together or took the form of a ridgepartially wrapped around the pocket. In somespecimens the villous processes were covered with amaterial, presumably the mucopolysaccharidesbetween outer receptors and the pigment epithelium.This material appeared as a plate with spaces for thereceptors (Fig. 3).The site of the zonula occludens was visible as a

band about 500 nm wide encircling each pigment cellat the junction between the apex and the sides (Fig.4). In most specimens this band was a flat outpouchingof the cell membrane at the sides of the pigment cell.In other specimens this band appeared to shrink fromprocessing more than the sides of the cell, whichsometimes bulged above the band.The zonula adherens and the maculae adherentes

were not visible on the sides ofthe pigment epitheliumby scanning electron microscopy. The smooth cellmembrane on the sides of the pigment epitheliumbulged over the melanin granules (Fig. 1).The basal surface of the pigment epithelium was

generally not visible from the outside. In onespecimen a sheet of remaining pigment epitheliumwas detached from the basal lamina and curled

Fig. 4 Pigment epithelium with aband at site ofzonula occludens(ZO) surrounding each cell atjunction between sides and apex.The sides show some deteriorationfrom exposure to trypsin. (x 3117).

19


http://bjo.bmj.com

/B

r J Ophthalm


nloaded from

http://bjo.bmj.com/

Michael H. Goldbaum and Kendall Madden

t r

Fig. 5 Sheet ofpigment epithelium that has peeled upfrombasal lamina. An amorphous material coats outer surface ofpigment epithelium (OSR). Some basal infolding is visible(arrow). (x 2147).

Fig. 7 Remnants ofbasalinfoldings attached by attachmentplaques (arrow) to pigmentepithelium. (x 10286).

Fig. 6 Partially digestedpigment epithelium. Remnantbasal infoldings (BI) on the basal laminashow that adhesionbetween basal lamina and basal infolding was stronger thanintegrity of cell in this eye. (x 5965).

20


http://bjo.bmj.com

/B

r J Ophthalm


nloaded from

http://bjo.bmj.com/


Fig. 8 Basal lamina peeling awayfrom inner collagenouszone. At low power basal lamina appears like a plasticmembrane. (x 2684).

Fig. 9 Basal lamina athigh magnification appears granularor velvety. (x 14316).

Fig. 10 Detachment ofpigmentepithelium (RPE) exposing outersurface ofbasal lamina (BL) muchin the manner ofa clinical retinalpigment epithelial detachment.Note amorphous material betweenpigment epithelium and basallamina (arrow). No attachmentsites are visible between basallamina and inner collagenous zone.In this specimen the retina remains,exposing outer segment (OS).(x 1496).

21


http://bjo.bmj.com

/B

r J Ophthalm


nloaded from

http://bjo.bmj.com/


Fig. 11 Where basal lamina is absent, the inner collagenouszone is visible, revealing tight interweaving ofcollagenfibres.The fibres occasionally appear joined together lengthwise(arrow). (x 5368).

inwards. An amorphous material adhered to theexternal surface, leaving clefts where basal infoldingwas present (Fig. 5).

PIGMENT EPITHELIUM ADHESIONS TO BRUCH'SMEMBRANEIn many specimens the adhesion of the basal cellmembrane to the basal lamina of Bruch's membranewas stronger than the integrity of the cell, leavingremnant basal cell membrane on the basal lamina.The internal aspect of this remnant membraneshowed basal infolding (Fig. 6). Transmission electronmicroscopy of the same piece showed attachmentplaques of the flat parts of the cell basal membrane tothe basal lamina (Fig. 7).Broad expanses of the basal lamina were com-

pletely bared following enzymatic digestion andirrigation. The inner surface of the basal lamina wassmooth and showed no evidence of the location ofattachment plaques of pigment epithelium (Figs. 8and 9).

BRUCH'S MEMBRANEFour of the 5 layers of Bruch's membrane were

O.pm

Fig. 12 With high magnification thefibres ofthe innercollagenous zone appear to have an external representationofbanding. (x 29825).

observed by scanning electron microscopy. Theprocess of enzymatic digestion and irrigation exposedmost of Bruch's membrane. 25 to 50% of the basallamina was lost from each specimen, allowing easyvisualisation of the inner collagenous zone. The restof Bruch's membrane generally remained intact. Theshrinkage produced by the critical-point processingcaused the edges of some specimens of Bruch'smembrane to curl inwards, allowing visualisation ofthe elastic layer and the outer collagenous layer. Thebasal lamina of the choriocapillaris endothelium wasnot seen.

Basal lamina of the pigment epithelium. By trans-mission electron microscopy the basal lamina showedas a straight, finely-granular membrane 40 to 100 nmthick (Fig. 1). Its appearance by transmission electronmicroscopy did not prepare one for its appearance byscanning electron microscopy. With moderate magni-fication the basal lamina appeared like a smooth thinplastic membrane (Fig. 8). At high magnification, themembrane took on a more granular appearance (Fig.9). Both surfaces were smooth. There was no visibleevidence on the inner surfacewhere pigment epithelialattachments were located. There was no evidence of

22


http://bjo.bmj.com

/B

r J Ophthalm


nloaded from

http://bjo.bmj.com/


Fig. 13 Elastic lamina with some collagenfibres lying on Fig. 14 Curling up ofpart ofBruch's membrane hasinner surface. Some ofinner collagen fibres are inserted into exposed outer collagenous layer. The collagenfibres are lesselastic layerandsomejoin outer collagenous layer bypassing tightly woven. (x 5276).through holes in elastic lamina. (x 8350).

insertion of collagen fibrils from the inner collagenouslayer into the basal lamina (Fig. 10). The basal laminatended to separate easily from the inner collagenouslayer; the attachment between the basal lamina andthe inner collagenous layer appeared to be much lessfirm than between the pigment epithelium and thebasal lamina (Figs. 6, 10).

Inner collagenous layer. At low magnification theinner collagenous layer appeared like velvet (Fig. 8).At moderate magnification the collagen fibres in theinner collagenous layer formed a tightly interwovenmembrane (Fig. 11). The ground substance betweenthe fibres was not visible. At high magnification thecollagen fibres appeared to have a uniform thicknessof about 70 nm (Fig. 12). There were transverse ridgeswith a periodicity of about 60 nm, perhaps repre-senting a surface manifestation of the banding seen inthese fibres by transmission electron microscopy.

Elastic layer. The elastic layer appeared to becomposed of coarse and fine fibres matted together bysome amorphous substance (Fig. 13). This layerformed an almost solid sheet with clefts and smallholes. Collagen fibres from the inner collagenous

layer appeared to be inserted into the elastic layer andalso to pass through holes in the elastic lamina to joinwith collagen fibres in the outer collagenous layers.

Outer collagenous layer. The collagen fibres in theouter collagenous layer were less dense, finer inappearance, and more loosely interwoven than thosein the inner collagenous layer (Fig. 14). The collagennetwork between the choriocapillaries appearedsimilar to the outer collagenous layer, with which itwas continuous (Fig. 15).

Discussion

The enzymatic loosening of the pigment epitheliumallowed scanning electron microscopy of Bruch'smembrane. Some of the pigment epithelium did notwash away, and the remaining cells showed variableamounts of enzyme effect, ranging from not visible toseverely digested.The microvilli around receptor elements have pre-

viously been observed by transmission electronmicroscopy to mat together and form ramparts.9 Theridge we saw with scanning electron microscopy may

23

'Y "W:V. i..`--., ON".y-yR

N.R........i.M M PR

W.


http://bjo.bmj.com

/B

r J Ophthalm


nloaded from

http://bjo.bmj.com/


Fig. 15 Outer collagenous layer is continuous with collagenfibres between vessels in choroid. Split in Bruch's membranehas exposed choriocapillaris and collagen fibres between.(x 3517).

be the same feature. On the other hand there couldbe an enzyme artefact. Likewise the plate wesometimes observed between pigment epithelium androds and cones could be mucopolysaccharides orcould be an enzyme artefact. The zonula occludenswas a surface feature visible as a band round the sidesof the cell near the apex.2 13 We believe the zonulaoccludens has not previously been described as avisible surface feature of the retinal pigment epi-thelium. It functions as an outer blood-retina barrierto large molecules. 14-6 The sides of the pigmentepithelium covered the pigment granules and wereotherwise smooth; attachment plaques at the sides

Fig. 16 Artist's representation ofpigment epithelium andBruch's membrane. Microvilli extendfrom apical surfaceofpigment epithelium, occasionally forming pockets for rodouter segment. The zonula occludens barrier surrounds eachcell, separating the side membrane and the apical surface.The basal lamina ofthe pigment epithelium is smooth like athin plastic membrane. Basal infoldings adhere to basallamina, which is not tightly attached to the inner collagenouszone. Inner collagenousfibres are more tightly woven thanouter collagenous.fibres. Elastic lamina forms a plate withholes between the 2 collagenous zones.

24


http://bjo.bmj.com

/B

r J Ophthalm


nloaded from

http://bjo.bmj.com/


of the pigment epithelium'7 did not produce visiblechanges on the surface.The adhesion between retinal pigment epithelium

and the basal lamina was stronger than between thebasal lamina and the collagenous layer. This latterjunction is also the site of separation for the clinicalcondition of retinal pigment epithelial detachment.Attachment plaques at the basal layer of the pigmentepithelium were seen in transmission electronmicroscopy, but were not visible as surface featuresby scanning electron microscopy. The amorphousmaterial observed on the outer surface of the cellbasal membrane (Fig. 10) may be involved in theadhesion to the basal lamina. There was no visiblemechancal attachment between the smooth outersurface 6f the basal lamina and the rest of Bruch'smembrane (Fig. 10).The tightly woven collagen fibres of the inner

collagenous layer and the dense elastic layer by theirappearance can be surmised to act as a flexible yetstable platform in 2 dimensions to support the retina,much as the plastic film base acts as a support for theemulsion in photographic film. Another function thatcan be hypothesised is that these layers act as amechnical barrier that keeps the choriocapillariesaway from the retina. Fig. 16 represents an artist'sconcept of the composite findings of scanning andtransmission electron microscopy.Dale Deg assisted in devising techniques to expose the layers inBruch's membrane.This work was supported in part by grant NIH EY 02434 and VA

grant MRIS 3192.

References

I Bruch KLW. Untersuchungen zur Kenntnis des KornigenPigments der Wirbeltier. Zurich: Diss, 1844.

2 Wolfrum M. Beitrage zur Anatomie und Histologie der Aderhautbeim Menschen und bei hoheren Wirbeltieren. Albrecht vonGraefes Arch Klin Ophthalmol 1908; 67: 307-59.

3 van den Hoof A. Het elektronen-microscopische beeld van hetmembraan van Bruch. Ned TijdschrGeneeskd 1954;98:2869-70.

4 Verin P, Gendre P, Le Rebeller M. Bruch's membrane and itsconnection with the choriocapillary. Arch Ophtalmol (Paris)1969; 29:123-34.

5 Sumita R. The fine structure of Bruch's membrane of the humanchoroid as revealed by electron microscopy. J Electron Microsc(Tokyo) 1961; 10: 111-8.

6 Nakaizumi Y. The ultrastructure of Bruch's membrane in human.monkey, rabbit, guinea pig, and rat eyes. Arch Ophthalmol 1964;72: 380-7.

7 Nakaizumi Y, Hogan MJ, Feeney L. The ultrastructure ofBruch'smembrane. III. The macular area of the human eye. ArchOphthalmol 1964; 72: 395-400.

8 Hogan MJ. Electron microscopy of Bruch's membrane. TransAm Acad Ophthalmol Otolaryngol 1965; 69: 683-90.

9 Spitznas M. The fine structure of the chorioretinal border tissuesof the adult human eye. Adv Ophthalmol 1974; 28: 78-174.

10 Wickham MG, Worthen DM. Correlation of scanning and trans-mission electron microscopy on the same tissue sample. StainTechnol 1973; 48: 63-8.

11 Sakuwagara M, Kuwabara T. The pigment epithelium of themonkey. Topographic study by scanning and transmissionelectron microscopy. Arch Ophthalmol 1976; 94: 285-92.

12 Angelucci A. Histologische Untersuchungen uber das retinalePigment Epithel der Wirbeltiere. Arch Physiol Lpz 1878: 358-86.

13 Verhoeff FH. A hitherto undescribed membrane of the eye andits significance. R Lond Ophthalmic Hosp Rep 1903; 15: 309-19.

14 Shiose Y. Electron microscopic studies on blood-retinal andblood aqueous barriers. Jpn J Ophthalmol 1970; 14: 73-87.

15 Peyman GA, Spitznas M, Straatsma BR. Peroxidase diffusion inthe normal and photocoagulated retina. Invest Ophthalmol VisualSci 1971; 10: 181-7.

16 Peyman GA, Spitznas M, Straatsma BR. Chorioretinal diffusionof peroxidase before and after photocoagulation. InvestOphthalmol Visual Sci 1971; 10: 487-95.

17 Farquhar MG, Palade GE. Junctional complexes in variousepithelia. J Cell Biol 1963; 17: 375-412.

25-


http://bjo.bmj.com

/B

r J Ophthalm


nloaded from

http://bjo.bmj.com/

a perspective on bruch's membrane and the retinalpigment ... · pigment epithelium...

Documents