British Journal of Ophthalmology 1995; 79: 76-80

Clinical pathology and retinal vascular structure inthe Bardet-Biedl syndrome

Toke Bek, Thomas Rosenberg

AbstractA comparative study of clinical pathologyand retinal vascular structure is describedas studied by vascular casting in an eye ofa patient with the Bardet-Biedl syndrome.At the time of examination the eye hadbeen almost blind for at least 4 years. Thehistopathological examination showed alargely uniform loss of the outer retinallayers. The gross pathological examina-tion of the cast ocular fundus showedthree distinct zones, an inner zone insidethe temporal vascular arcades whereretinal vessels had been cast, a midperipheral zone with bone spicules, and aperipheral zone with neither cast vesselsnor bone spicules. The findings are dis-cussed in relation to possible patho-physiological mechanisms involved in thedevelopment of retinal dystrophy in theBardet-Biedi syndrome.(Br_J Ophthalmol 1995; 79: 76-80)

Department ofOphthalmology, ArhusUniversity Hospital,DK-8000 Arhus C,DenmarkT Bek

National Eye Clinic forthe Visually Impaired,1 Rymarksvej,DK-2900 Hellerup,DenmarkT Rosenberg

Correspondence to:Toke Bek, MD, Departmentof Ophthalmology,University of Arhus, ArhusKommunehospital, DK-8000 Arhus C, Denmark.

Accepted for publication5 September 1994

The Bardet-Biedl syndrome (BBS) is a heredi-tary autosomal recessive disease characterisedby retinal dystrophy, mental retardation, poly-dactyly, obesity, and hypogenitalism. Internalorgans may also be affected, and early deathmay result from renal failure. One or more ofthe clinical features characterising the syn-drome may be absent, but retinal dystrophy isa consistent finding.1 2 Retinal dystrophybecomes clinically manifest in early childhood,with progressive loss of visual function,leading to severe visual disability in earlyadolescence.2-5 Morphologically, the retinaldystrophy is characterised by a bull's eyeappearance in the macula, and in the latestages morphological signs of retinitis pigmen-tosa develop, with bony spicules in the midperipheral retina.The pathogenesis of retinal dystrophy in

BBS is unknown. Histopathological studies ofeyes at different stages of the disease point to a

series of events with primary degeneration ofphotoreceptors and secondary involvement ofthe other retinal layers including the retinalpigment epithelium.69 Clinically, the progres-sive loss of retinal tissue is accompanied byophthalmoscopically visible attenuation of theretinal vessels. However, more detailedevidence about the distribution and structureof retinal vessels in BBS is yet unreported.

This paper reports a comparative study ofclinical pathology and retinal vascular struc-ture as studied by vascular casting in a case ofBBS. The findings are discussed in relation to

adaptive properties of retinal vascular structureand in relation to possible pathophysiologicalmechanisms involved in the development ofretinal dystrophy in BBS.

Case reportThe patient was a female, first examined by anophthalmologist at the age of 11 years and 9months. At that time she reportedly had beennightblind for several years, and visual acuityhad gradually decreased during the 6 monthspreceding the examination. The ophthalmicexamination showed visual acuity of 6/36 inboth eyes. Comeal sensibility, ocular motility,and pupil reaction were normal. Ophthal-moscopy showed optic nerve atrophy andthinned retinal arterioles.At the age of 13 years and 6 months the

patient was seen at the Danish National EyeClinic for the Visually Impaired where the diag-nosis of retinitis pigmentosa secondary to theBardet-Biedl syndrome was established.Parental consanguinity was denied and norwere any relatives known to have had thedisease. The birth of the patient had beennormal. She had attended a class for retardedchildren and had done reasonably well in spiteof the fact that reading was only possible in verybright light. Physical examination showed:height 144 cm, weight 71 6 kg, and severeobesity of head, trunk, and limbs. Physicaldevelopment lagged behind chronological age,but mental development was within the normalrange. x Ray examination showed normal sellaturcica and brachydactyly ofhands and feet, butno supemumerous fingers or toes. Ophthalmicexamination showed VA right eye 6/24 + 1 0 Dcyl ax 90°, VA left eye 6/24 +2-0 D cyl ax 900.There was slight nystagmus. Visual field exami-nation on an arc perimeter with white targets10/300 and 3/300 was normal in both eyes.Ophthalmoscopy showed pale optic nerveheads, thin retinal vessels, bone spicules in theretinal mid periphery, and clearly visiblechoroidal vessels in the retinal periphery.The patient was examined regularly over

the next 6 years, during which time the visualacuity gradually decreased, the visual fieldnarrowed, and the mid peripheral bonespicules increased. At the age of 14 years and 6months reading capability had been almostlost, even in very bright light. At the age of 17years visual acuity was VA right eye <6/36+10 D sph +1-0 D cyl ax 900, and VA left eye<6/36 +1I0 D sph, +1I0 D cyl ax 900. At theage of 21 years visual acuity had decreased toVA right eye 6/60, VA left eye 1/60.

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Clinical pathology and retinal vascular structure in the Bardet-Biedl syndrome

Figure 1 The central retinal zone with cast vessels. The optic nerve head (ONH) is seento the upper right, surrounded by extensive artefactual extravasation ofcasting material.Similar extravasation of casting material is seen around thefovea in the centre of thefigure. Vascular anastomoses between the upper and the lower temporal arcade are seentemporal from the foveal area. Bar= 1 mm.

The next, and last, recorded ophthalmicexamination is from the patient's localhospital, performed at the age of 56 yearsduring admittance for chronic pyelonephritis.At that time the visual acuity of both eyes waslight perception with uncertain projectionsense. There were nystagmoid eye movements,but the pupils reacted normally to light andconvergence. Slit-lamp examination showedbilateral posterior subcapsular cataract, butotherwise appeared normal. Ophthalmoscopyshowed pronounced synchysis scintillans onthe right eye. Both eyes had maximally paleoptic nerve heads, thinned retinal vessels, andperipheral hyperpigmentation indicative ofretinitis pigmentosa.

During the following years, progressive renalfailure with rising serum creatinine and serumcarbamide developed, and the patient eventu-ally died from uraemia at the age of 60 years 6months. On regular admissions to hospitalduring the progression of renal failure theblood pressure never exceeded 135/80 mm Hg.

Materials and methods

TISSUE MATERIALThe left eye of the patient was donated to thecornea bank at the Department of Oph-thalmology, Arhus University Hospital. Theeye was small, but otherwise it appearedmacroscopically normal. The cornea was clear,had normal endothelial density, and displayedno pathology that disqualified it from beingused as a donor graft. The pupil was round, theiris appeared normal, and the lens was catarac-tous. The axial distance from the ora serrata tothe macular retina could be measured with aslide gauge after the cornea, including acircular scleral rim, had been removed bytrepanation. This estimate of the axial lengthofthe eye was found to be 13 mm. Postmortemtime at fixation was 45 hours. During all

preparation procedures, the retina was firmlyadherent to the underlying choroid and dis-played no tendency to detach.

CASTING PROCEDUREThe casting procedure was a slight modifica-tion of the procedure described in Bek andJensen.'0 Casting was technically successful,but increased resistance was met at the injec-tion of casting material into the retinal vessels.After the casting procedure, the eye wasimmersed in a solution of equal parts 25%glutaraldehyde and 4% formalin for 20 hours.The iris and the lens were subsequentlyremoved, and a liquified vitreous decanted.The hollow eyeball was dehydrated in ethanol,and the remaining denatured vitreous and theposterior hyaloid membrane were removedwith a swab. The inner aspect of the eyeballwas documented by photography on TMY400 black/white film (Kodak) through one ofthe oculars of a Zeiss SV 11 stereo micro-scope.

HISTOPATHOLOGYTwo horizontal slices of the eyeball wereexcised for histological examination, located atthe level around the upper temporal vasculararcade and around the fovea, respectively.Histological sections were made parallel withthe cut surface of-these slices, and thus werecontinuous from the nasal periphery, throughthe macula to the temporal periphery.Successive sections were stained with haema-toxylin and eosin, periodic acid Schiff withhaematoxylin and toluidine blue.

Results

GROSS PATHOLOGY OF THE OCULARBACKGROUNDOn the basis of the morphological appearance,the ocular fundus could be divided into threeconcentrical zones that differed markedly fromnormal eyes of similar age.'0The central zone consisted of the area where

retinal vessels had been cast to appear white(Fig 1). This zone comprised the macular areaincluding the temporal arcades, extendingnasally one disc diameter from the optic nervehead. The cast vessels appeared thin and dis-played markedly decreased density. Thenormal three layered arrangement of capillariesin the macular area was absent, and onlyoccasionally did the capillaries form two layers,mainly close to the optic nerve head where themost proximal part of the radial peripapillarycapillary network was preserved. Along thetemporal raphe, extensive communicationswere seen between the vessels originating fromthe upper and the lower temporal arcade. Inthe periphery of the zone, the cast vesselsended in terminal single layered arcades(Figs 1, 2). These arcades and other bordervessels were continuous, with no abruptclosures towards the retinal mid periphery.However, in a transition zone, the cast vessels

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Figure 2 The temporal border of the central cast vessel area, with anastomosing terminalarcades from the upper (U) and the lower (L) temporal arcade. A few retinal vessels areseen to be ensheathed by hyperpigmented material. Bar=250 ,um.

were discontinuously ensheathed with hyper-pigmented spicules that branched off to con-tinue into the retinal mid periphery in radiatinglines, as if representing sites of former vessels(Fig 3). A notable artefact was diffuse intra-retinal extravasation of casting material at theoptic disc so that its margins had becometotally obscured (Fig 1). A similar pattem ofdiffuse extravasation of casting material waspresent in an area around the foveal avascularzone, thus obscuring the vessels in this area,including possible gross pathological signs ofmacular degeneration.The mid peripheral zone was characterised

by an abnormal accumulation of hyperpig-mented bone spicules. This zone was locatedimmediately peripheral from the central zone,as a concentric ring area with a constant widthof approximately 6 mm. Nasally, this zonealmost reached the ora serrata, whereas abroader zone separated it from this margin onthe temporal side. The bone spicules could belocalised in two distinct layers. The spicules of

the inner layers were generally long and thin,and often constituted extensions of cast vesselsfrom the central retinal zone (Fig 3). Thedeeper lying spicules generally were broaderand coarser, and showed no visible relation toother anatomical structures.The peripheral zone was greyish looking with

sparse bone spicules. The width of this zonevaried from approximately 2 mm nasally to7 mm temporally. The few bone spicules herewere predominantly located in deeper layers,often bounding rounded stamped out lesionsvarying in size from 300 to 800 ,um (Fig 4).

Collateral filling from the injection catheterin the central retinal artery into choroidalvessels was seen close to the optic disc, and ina few vessels that radiated more distantlytowards the retinal periphery.

HISTOPATHOLOGICAL FINDINGSThe histological sections showed a total lack ofphotoreceptors in all retinal areas. In thecentral and the mid peripheral retinal area thepigment epithelium was discontinuous withthe cells arranged in separated islands, whereasthis cell layer was continuous in most of theperiphery. At places where pigment epithelialcells were present, these appeared atrophic andthe outer retina had been detached from them,whereas the outer retina adhered to the under-lying choroid where the pigment epithelial cellswere absent (Fig 5). Bruch's membrane wasuniformly thickened at all eccentricities, andno sign of the choriocapillaris could be recog-nised. The choroid appeared fibrotic, butlarger choroidal vessels were present. In theinner retina, the inner nuclear layer and fewganglion cell bodies could be distinguished.The cell density of these layers was consider-ably decreased, and gliosis was prominent.Muller cell processes traversing the innerretina were clearly seen in most of the sections.Larger retinal and choroidal vessels displayedthickening of the walls but no specific hyper-tensive changes could be recognised.

Focal black areas appeared in two forms inthe inner retina. One form consisted of castingmaterial, either as well delimited round areasofblack material representing shrunken castingmaterial inside retinal vessels (Fig 5) or asirregular extravasated lumps. Cast vessels werealmost exclusively seen around the inner plexi-form layer and the ganglion cell layer, whereasa very few vascular loops were seen to take acourse down towards the outer plexiform layer.In the macular area extravasated castingmaterial was seen inside cystoid spaces in theinner retina. The other form of black materialappeared as grainy aggregates of pigment(Fig 6), predominantly associated with retinalvessels, but also occurring in irregular lumpselsewhere in the retina (Fig 5). The blackgrains seen at the peripheral aspect of thewalls of larger retinal vessels occupied thewhole circumference or parts of it (Fig 6). Insome instances, thickened circumferentialhyperpigmentation protruded to narrow thevascular lumen appreciably. Occasionally, thevascular lumen was totally obliterated by the

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79Clinical pathology and retinal vascular structure in the Bardet-Biedl syndrome

Figure 3 Retinal vessels ensheathed with hyperpigmentation in the border zone betweenthe retinal centre and mid periphery. Bar=250 am.

hyperpigmentation, and occasionally PASpositive material occluded a lumen narrowedby hyperpigmented material.The only conspicuous hyperpigmentation in

the deeper retinal layers was patchy hyper-plasia of the retinal pigment epithelium occur-

ring in the retinal mid periphery and peripheryprobably accounting for the deeper lyinghyperpigmented spicules seen in the ocularbackground.

DiscussionIt is a matter of dispute which signs in the clin-ical spectrum of the Bardet-Biedl syndromeare necessary and sufficient to establish thediagnosis in borderline cases, since some

phenotypical variation exists. In the presentcase the phenotype was typical of BBS, despitethe absence of polydactyly. Additionally, thediagnosis was indirectly supported by broadhands with short fingers, early involvement ofthe central retina, and renal failure.

Current evidence suggests that the primaryevent in the pathogenesis of BBS is degenera-tive loss of retinal photoreceptors, withsecondary involvement of the inner retinal

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Figure 4 The transition zone from the retinal mid periphery with many bony spicules(bottom) to the greyish looking periphery with sparse spicules (top). Deep stamped outlesions are seen (arrows). Bar= 750 ,um.

layers and the retinal pigment epithelium. Thisevidence is supported by histopathologicalstudies,6 7 11 and by electrophysiological find-ings of reduced or absent ERG responses fromretinal photoreceptors, and frequently occur-ring EOG changes indicating damage to thepigment epithelium. 12-14 The findings of thepresent study are in accordance with thisevidence. As judged from the clinical historythe examined eye had reached a ratheradvanced stage of retinal dystrophy, and thiscould be confirmed by the histological findingsof pronounced degeneration in all areas of theretina and the choroid. The clinical historywith normal blood pressure makes it unlikelythat the vascular pathology should be due toarterial hypertension.A striking feature of the examined eye was

the lack of cast vessels in the retinal midperiphery displaying bone spicules characteris-tic of retinal dystrophy. The bone spiculesprobably represent melanin from retinalpigment epithelial cells migrated into the innerretina. This pattern of pigment migration is aresponse common to various vascular, inflam-matory, and dystrophic disease processes.However, in retinitis pigmentosa the migratedpigment displays a characteristic ring shapeddistribution in the retinal mid periphery. It isgenerally assumed that this geographicalpredilection reflects the fact that photoreceptordegeneration begins in this area. However, itmight be anticipated that photoreceptor lossoutside the mid peripheral retinal zone wouldhave a similar potential for inducing pigmentepithelial migration as it would have in the midperipheral zone. The different responsepatterns in different retinal areas mightperhaps be due to some regional variation inretinal anatomy or physiology, the nature ofwhich is presently unknown.The generalised thinning of retinal vessels

seen ophthalmoscopically in retinitis pigmen-tosa could also be recognised in the cast retinalvessels of the eye examined here. An exactmeasure of vascular thickness would beinvalid, however, because of variable arte-factual shrinkage of the casting material. In theexamined case the transition from the centralarea with cast vessels to the mid peripheral areawith bone spicules was rather abrupt, with onlyfew border zone vessels both containing cast-ing material and being ensheathed with pig-ment granules. One might, therefore, suggestthat vascular occlusion was in some wayrelated to the accumulation of pigmentmaterial in and around retinal vessels. Theexact nature of such a relation remains to beelucidated, however.The loss of three dimensional structure of

vessels in the macular retina was seen histo-logically to be due to occlusion of the externalretinal capillary layers. This is in accordancewith the presumption that photoreceptordegeneration will mainly lead to decreasedmetabolic demand in outer retinal layers. It isnotable, however, that any vessels remained inthe macular retina at all. As judged from thehistological appearance, the degenerativeprocess of the retinal tissue had almost reached

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Figure 5 Haematoxylin and eosin stained section of the macular retina. Shrunken castingmaterial is seen inside several retinal vessels all localised in the same retinal layer (smallarrows). An area with pigment epithelial hyperplasia is seen at the junction towards thechoroid (large arrow). Bar= 100 ,um.

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Figure 6 Periodic acid Schiffand haematoxylin stained section of the retinal midperiphery. Retinal vessels are seen to be ensheathed with hyperpigmented granular materialprotruding into the vascular lumen in a varying degree. The remaining parts of the lumenare occluded by periodic acid Schiffpositive material. Bar= 100 ,um.

the same degree of severity in all retinal areas.

Therefore, the persistence of retinal vessels inthe macular area seems peculiar. This findingcould perhaps be due to the fact that the patho-physiological process involved in the remodel-ling ofretinal vascular structure had not reachedits final level, or that the remodelling processhad been unable to progress to a degree wheremacular blood flow was totally eliminated.

It is often assumed that the superior andinferior temporal retinal arcade vessels are endvessels, with no anastomoses connecting thetwo. The identification of such anastomosesis difficult in normals because of the densedistribution of retinal vessels along the tempo-ral raphe, especially in the macular area. How-ever, with the casting technique employed in

the present study, frequent anastomoses at thecapillary level have been found between theupper and the lower temporal arcade along thetemporal raphe in normals (unpublished).Thus, the anastomoses found between theupper and the lower temporal arcade in thepresent study may be the result of an adaptiveremodelling of anastomoses normally present,which have become recognisable because ofthe pronounced decrease in vascular density inthe macular area.The artefact of casting material extra-

vasating diffusely into the retinal tissuearound the optic nerve head is seen in normaleyes when the casting material is introducedinto the central retinal artery under highestpossible pressure. The presence of this arte-fact may therefore be due to increased flowresistance in the diminished retinal vascularbed of the cast eye. The extravasation of thecasting material around the fovea, however, ispeculiar and has not been experienced beforewith the employed casting technique. It ispossible that the perifoveal capillaries mayhave been especially vulnerable to the castingpressure transmitted to the capillary level.Such a vulnerability might perhaps be relatedto the cystoid degeneration noticed in thisarea histologically, and might be part of amacular dystrophy.

In conclusion, the present case report hasshown an abnormal distribution and structureof retinal vessels in an advanced case of theBardet-Biedl syndrome. The findings maycontribute to better understanding of adaptiveproperties of the retinal vasculature in retinaldystrophies.

The skilful assistance of technicians Birthe Olesen and EnaKristensen is gratefully acknowledged.

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