@Pergmnon

VisionRes., Vol.36, No. 14, pp.2015–2028,1996Copyright() 1996ElsevierScienceLtd.AUrightsreserved

0042-6989(95)00317-7 Printedin GreatBritain0042-6989/96$15.00 + 0.00

Immunohistochemical Study of Human OpticNerve Head AstrogliaALBERTO TRIVINO,* JOSE M. RAMfREZ, JUAN J. SALAZAR, ANA I. RAMfREZ,JULIAN GARCfA-SANCHEZ

Received 3 March 1995; in revisedform 2August 1995; infinalform 9 November 1995

Immunocytochemicallocalizationof glial fibrillaryacidicprotein(GFAP)has been used to studythe distributionof astrocytesand their morphologyin.sectionsof the optic nerve (ON) of humaneye.Althoughall ON regionspresentedGFAPimmunoreactivity,immunostainedtissuewas mostcommonin the posteriorprelaminarregion (PR) and least commonin the laminarregion (LR).Two shapesof astrocyteswere distinguished:thick and thin bodied astrocytes.Aatrocyteswiththickceil bodiesare locatedin the superllcialnervefiberlayer (SNFL),PR LR and retrolaminarregion(RR).Astrocyteswiththincellbodieswere foundin the SNFLandanteriorPR Sometimesthin bodiedastrocytespresentedanothershapewith a long processrunningparallelto the axonsand these were foundin the PR and LR In the SNFL the thin bodiedastrocytesaccompanytheaxonsandcontactthecapillariesderivedfromthecentralretinalartery.IntheanteriorPRthethinbodiedastrocyteswitha stellateshapelie overthe vesselsforminga sievethroughwhichthe axonspass.In the posteriorP~ the thickbodiedastrocyteaformglialtubesthatdirectaxonstowardstheLR. These astrocytesform a layer in the IX that lines the pores of the Iaminacribrosa andseparatesthe connectivesepta from the axon bundlesin the RR The limitingglial membranesseparatethe ON tissuesfromthea~acent tissuesand fromthe courseof the centralretinalarteryand are composedof manythickbodiedastrocytes.Copyright01996 ElsevierScienceLtd.

Human Optic nerve head GFAP Astrocytes Immunohistochemistry

INTRODUCTION

The optic nerve (ON) k fundamentally made up ofganglion cell axons, glial cells, blood vessels andmesodermictissue.Among neuroglia,astrocytesare veryimportant to ON function since they perform many tasksnecessary for axonal survival. Most importantly, theymaintain ionic equilibrium, regulate the neuronal meta-bolism, have a role in inducing the blood+ptic nervebarrier and participate in the scarring and repair processof the nervous system (Kimelberg & Ransom, 1986;Abbott et al., 1992).

Most studieson ON astrocyteshave been performedinexperimental animals especially focusing on their originand characteristics of the different macroglial cell lines(Raff et al., 1987;Miller et al., 1989;Raff, 1989;Skoff,1990).The first morphologicalstudies on ON astrocytesin humanswere performedwith silver techniques(Davis,1940; Liss, 1956; Welter, 1957), and reported a largevariety of astroglialmorphologiesalthough the differentauthors never agreed on what they had seen. The mostimportant observations in human ON astrocytes, made

Instituto de IrtvestigacionesOftalmo16gicas ‘Rarn6n Castroviejo’,Facultad de Medicina, Pab. VI, 4’ Planta. Univeraidad Complu-tense, 28.040Madrid, Spain.

*Towhom all correspondenceshouldbe addressed.

with electron microscopy, have established the ultra-stnmtural characteristics of the cells (Anderson et al.,1967;Anderson, 1969, 1970;Anderson & Hoyt, 1969).

Immunohistochemicalstudies on the astrocytes of thehuman ON are quite scarce, and even less have been doneusing the anti-GFAP staining technique which allowsselective cell labeling (Dahl et al., 1986). Studies usingthis technique have investigated astrocytes during thedevelopmentof the ON (Rhodes, 1982; Quistchke et al.,1985). This paucity of studies does not accord with thefunctional importance of astrocytes in ischemic vascularpathologies like ischemic optical neuropathy or glau-coma (Radius, 1987).

Astrocytes are the intermediariesbetween the vascularsystem and the axons, and during ischemia they undergoa number of biochemical and morphological modifica-tions that provoke axonal death (Flammer, 1994). Giventhe lack of anti-GFAP morphological studies and thefunctional importance of ON asbocytes in differentischemic pathologies, this paper has undertaken amorphological immunohistochemical study of healthyON astroglial cells that determines their distribution inthe different regions of the ON head.

MATERIALS&METHODS

Forty normal adult human eyes (age range: 20-402015

——.. . . . —..—.

2016 A. TRIVINOet al.

SNFL

anterior

postcriol

LR

RR

I

FIGURE1.Anti-GFAPsectionsshowingdifferentregionsof the humanON.(A): ONhead regions:superficialnerve fiber layer(SNFL),anterior prelaminar region (APR),posteriorprelaminarregion (PPR); laminar region(LR); retrolaminarregion (RR);and the different glial limiting membranes: Elschning’s limiting membrane (l), Kuhnt’s central meniscus (2), Kuhnt’sintermediary tissue (3), Jacoby’s tissue (4), Graefe’s peripheral mantle (5), and the prolongationof Kuhnt’scentral meniscus(6). (B): SNFL: Elschning’s limiting membrane (+). Parallel elongated astrocytes (>) lie below the limiting membrane;stellate astrocytessurroundbloodvessels (V). (C): PR:Anteriorprelaminarregion(APR):the astrocytesare arrayed in columns(*). posterior prelaminarregion(ppR): astrocytesform wide bands (amows).[C: choroid;CV:central retinal vessels; R: retina;S: sclera; V: vessel; VH: vitreoushumor;(A): longitudinalsections6 #m; (B,C): longitudinalsections 12pm; (A): PAP; (B,C):

immunofluorescence-FITC;(A): 63x; (B): 500x; (C): 156x.]

years), which had been enucleatedabout 2-4 hourspost-mortem for comeal transplantation,were obtained fromthe Spanish Eye Bank for this study. One to two hoursafter enucleation and corneal processing, the eyes werefixed with 4% paraformaldehyde in 0.1 M phosphatebuffered saline (PBS) at pH 7.4 for 4 hr at 4°C. Theposteriorsegmentwas separatedfrom the eyes to preparelongitudinal and transverse sections. The tissue wasdehydrated,embedded in paraffinand cut into 4,6,8, 12and 30 m thick sections.

Immunocytochemical procedure

For the peroxidaseantiperoxidasereactionwe used the

PR

PR

following procedures. The ONS were pretreated with0.390 hydrogen peroxide in PBS for 30 min at roomtemperature before washing in PBS (3 x 10 rein). Theywere then incubated for 3 hr at 4°C in 10?ZOnormal goatserum (NGS) (Sigma, U.S.A.) and 0.2Y0Triton X-1OO(Merck, Germany) diluted in PBS. ONSwere incubatedin monoclinal mouse antibody directed against GFAP(clone GA-5) (Sigma, U.S.A.) in a 1/250 dilution at 4°Cfor 12 hr and then washed in PBS (3 x 10 rein). This wasfollowed by incubation at 4°C for 6 hr with theimmunoglobulin fraction from goat antimouse serum(Sigma) diluted 1/100 before washing in PBS (3 x 10rein) followed. After that, they were again incubated at

IMMUNOHISTOCHEMICALSTUDYOF HUMANOPTIC NERVE HEAD ASTROGLIA 2017

FIGURE2. Morphologyanddistributionof anti-GFAPlabeledONaatrocytes.(A): LR:The numberof astrocytes(>) decreasesand they only cover the collagenous tissue (CT) of the cribriform plates. (B): A thin bodied aatrocyte (>) with elongatedmorphology extends a process (+) towards a capillary (CAP) in the SNFL. (C): Thin bodied astrocytes with stellatemorphology(>) located in the anterior PR. (D,E): Thin bodied astrocytes (>) with a single process parallel to the axon andradiating processes in the anterior PR (D) and the posterior PR (E). [CT: connective tissue; VH: vitreous humor; (A,E):longitudinalsection30 pm; (B,C,D):longitudinalsection 12pm; (A, C, F, G): PAP;@,E): PAPcontrastwith hematoxylin;(B):

immunofluoresccnce-FITC;(A): Nomarskioptics; (A,B): 500x; (C): 400x; (D): 156x; (E): 312x.]

4°C for 6 hr with mouse peroxidase antiperoxidasecomplex (PAP) (Sigma) diluted 1/500and rinsed in PBS(3 x 10 rein). The ONS were treated with 0.03%diaminobenzidinetetrahydrochloride(DAB) (Sigma) inPBS for 5 rein, followedby 0.03%DAB/O.01%hydrogenperoxide in PBS for an additional 7 min. After finalrinsing in PBS (3 x 10 rein), the ONSwere dehydratedina series of alcohols, cleared in xylene and mounted inDepeX (Serva, Germany). In addition to the PAPtechnique, indirect immunofluorescence was used insome cases. In this technique the GFAP antiserum(diluted 1/100) was followed by goat antimouse anti-bodies conjugated to fluorescein-isothiocyanate(FITC)(Sigma) diluted 1/20for 12 hr. In this case the ONSweremounted without dehydration in Citifluor (Agar, U.K.).All antibodydilutionswere carried out in 0.2% Triton X-100, 190 NGS in 0.1 M PBS at pH 7.4. Controls weretreated in the sameway as aboveexceptthat incubationinthe primary antibody solutionwas eliminated to demon-strate that the secondary antibodies reacted only withtheir respective primary antibody. Some contrast stainswere also donewith hematoxylinand picroindigocarminein the PAP-stained ONS.

RESULTS

For convenience in describing the location anddistributionof the astrocytes, the ON head was dividedinto several regions. Astrocyte architecture variedconsiderably between locations and the ON head couldbe divided into at least four regionson this basis (Fig. 1):the superficialnerve fiber layer (SNFL), the prelaminarregion (PR) the laminar region (LR) and the retrolaminarregion (RR).

LOCATIONOFGFAPACTIVITYINTHEOPTICNERVEHEAB

All the regions of the ON head are GFAP+ onlongitudinal sections [Fig. l(A)]. The most intenseimmunoreactivity is located at the level of the tissuesor membranes that isolate the ON from the vitreoushumor (VH), the retina, choroid, and the piamater; thesecmrespond respectively to Elschning’s limiting mem-brane, Kuhnt’s central meniscus, Kuhnt’s intermediarytissue, Jacoby’s tissue and Graefe’s peripheral mantle[Fig. l(A)]. Intense immunoreactivity also covers thecentral vessels (CV,)(the central retinal artery and vein)

2018 A. TRIVINO et al.

SCHEME 1. Three-dimensionaldrawing of the human ON head showing the glioarchitecture of the astrocytes around opticnerve fibers. (C: choroid; R: retina; S: sclera). 1. Superficial nerve fiher layer; 2. Anterior prelaminar region; 3. Posteriorprelaminar region; 4. Laminar region; 5. Retrolaminarregion.

in a prolongationof Kuhnt’s central meniscus. Immuno-reactivity varies from the SNFL to the RR in the rest ofthe ON [Fig. l(A)]. At the level of the SNFL immuno-reactivity is strong and a narrow GFAP+ band is visiblebelow Elschning’slimiting membrane [Fig. l(B)].

GFAP immunoreactivity is also strong in the PRalthough the proportion of labeled tissue varies dividingthe region into two areas: the anterior PR and theposterior PR. The anterior PR corresponds to the ONzone that is surroundedby retinal tissue.Columnsor linesof labeled tissuealternatewith wider less stronglystainedbands in this area [Fig. l(C)]. The posterior PR issurrounded by the choroid. The total amount of labeledtissue increases in this zone forming wide bands thatalternatewith narrower spaces in which GFAP+ tissue isscarce [Fig. l(C)]. The centerof this zone sinksto adaptitto the LR, so the middleof the posteriorPR is deeperoverthe LR than at the edges which abut on Jacoby’slimitingmembrane.

Collagenousscleral tissue enters the ON at the LR andforms the cribriform plates. This sclera is not GFAP+and the amountof labeledtissuedecreasesmarkedly [Fig.l(A)] although immunoreaction is still intense butrestricted to fine lines bordering the collagenous tissue

in the lamina cribosa [Fig. 2(A)]. The RR presents highGFAP immunoreactivity;the strongly labeled tissue liesin narrow lines outliningwider bands with a less intenseGFAP+ tissue [Fig. l(A)].

The various locationsof GFAP activity in the differentON regions are summarized in a three-dimensionalreconstructionshown in Scheme 1.

ASTROCYTEMORPHOLOGY

Human ON astrocytes present one of two basic bodycharacteristics: astrocytes with thick cell bodies or thincell bodies.

Thin bodied astrocytes

A highly GFAP+ small oval cell body of thin bodiedcells has an ovoid nucleus. Normally two to four verylong primary processes leave this perikaryon; they canfork into thinner secondaryprocesses,but very thin shortprocesses can also extend radially from the cell body[Fig.2(B,C)].These cells are located in the SNFLand theanterior PR. The morphology of these astrocytes variesfrom elongated to stellate. Elongated astrocytes extendprimary processesfrom both poles of the cell body in thedirection of the ganglion cell axons [Fig. 2(B); Scheme

IMMUNOHISTOCHEMICALSTUDYOF HUMAN OPTIC NERVE HEAD ASTROGLIA 2019

B

SCHEME2. Drawingof thinbodiedastrocytemorphology.A: anteriorPR stellate thin bodied astrocyte; B: SNPL elongated thin bodiedastrocyte; C: Thin bodied astrocyte with short radial processes and a

single long process runningparallel to the axons.

2]. In stellate astrocytes the primary processes extendradially out from the cell body [Fig. 2(C); Scheme 2].However, this group of thin bodied astrocytes alsopresents another morphologicalvariety. Seven to eightthin short processes can extend radially from the cellbody as well as a single, long straight process whichfollows a trajectory parallel to that of the ganglion cellaxons [Fig. 2(D,E); Scheme 2]. These cells are found inthe PR and LR lying parallel to the longitudinalaxis ofthe ON.

Thick bodied astrocytes

Thick bodied astrocytes with a prominent ovoidnucleusare even more stronglyGFAP+ than thin bodiedcells. Normally six to eight primary processes extendmore or less radiall~rfrom the cell body, dependingon itslocation within the ON. These primary processes candivide into thinner secondary processes. Short thinprocesses may also leave the cell body, [Fig. 3(A,B)].These astrocytesare distributedthroughoutthe ON from

SCHEME 3. Drawing of thick bodied astrocyte morphology A:limitingmembraneaatrocyte;B: posteriorPR and LR astrocyte;C: RR

astrocyte.

the SNFL to the RR and their morphology seems tochangewith the characteristicsof the surroundingregion(Scheme 3). They form the astroglial tissue of theposterior PR [Fig. 2(E)], LR [Fig. 2(A)] and RR [Fig.3(A)], make up the different glial limiting membranes[Fig. 3(B)], and sheath the central retinal vessels [Fig.3(c)].

DISTRIBUTIONOFOPTICNERVEHEADASTROCYTES:GLL4LARCHITECTURE

Superficial nerve jiber layer

The astrocytesmakeup a glial limiting membrane thatseparates the outermost ON tissue from the vitreoushumor; this is Elschning’s limiting membrane. Themembrane is made up of 2–3 layers of thick bodiedastrocytes and the cell bodies form groups of 4-5 incertain areas [Figs 1(A,B), 2(B)]. Lying parallel to eachother, theirprocessesform a glial net that is parallel to thevitreous humour [Fig. 3(D)]. The astrocytes in theinnermost layer of this limiting membrane also extendprocessestowardsthe interiorof the SNFL, givingthem astellate appearance. These processes can contact withblood vessels [Fig. 3(E)].

This layer bulges up towards the vitreous humor overthe blood vessels leaving the ON in the direction of theretina. The cell bodies of these astrocytesgroup on each

2020 A. TRIVINOet al.

FIGURE 3. Morphology and distribution of thick bodied astrocytes. (A,B) Thick bodied astrocyte (>) with a stellatemorphologylocated in the RR (A) and in Graefe’s peripheral mantle (B). (C) Thick bodied astrocytes surroundinga centralretinal artery (CRA). (D,E): Elschning’s limiting membrane (D) The processes of the thick bodied astrocyte net isolate thevitreous humor (VH) from the ON; (E) The thick bodied astrocytes ($+)grouped on each side of the vessel emit processestowards the vascular wall (V) giving the appearanceof a spindle and extend processes towards the interior of the SNFL(-+).(F) Interfascicular (+) and intrafascicular astrocytes in the RR. Intrafascicular astrocyte processes (A) extending towardsseptum walls (CT) and capillaries (CAP). [PR: posterior prelaminar region; (A,C,D,E): longitudinal section 30 #m; (B,F):transverse section 4 pm; (A,C,D,E):PAP; (B,F): PAPcontrast stain with hematoxylin,(A,C,D):Nomarskioptic; (A,D): 500x;

(B): 625X; (C): 312x; (E,F): 400x.]

side of the widest vessels and emit processes that totallyenvelop the vascularwall, giving them the appearanceofspindles [Fig. 3(B)]. Thinner blood vessels are onlysurroundedby glialprocesses.The parallelpositionof theprocesses in this area is less obvious due to thedisorganized course of the blood vessels. Thick bodiedastrocytesare concentratedin the center of the optic diskand form a wedge that covers the central vessels andcorresponds to Kuhnt’s meniscus [Fig. l(A)]. Thisastroglialsheath surroundsthe central vessels from theirentry point to and along the course of their path throughthe ON [Fig. 3(C)].

Thin bodied astrocytes are also found in the SNFL.They accompanythe ganglioncell axons from the retina

and may contact the capillaries derived from the centralvascular system [Fig. 2(B)]. The astrocytes underElschning’s limiting membrane lie parallel to themembrane and present an elongated morphology [Figsl(B), 2(B)]. In the most internal and central zones of theSNFL the astrocytes appear stellate because theirprocesses are directed towards many blood vessels inthis zone [Fig. l(B)].

These results are summarized in a three-dimensionalreconstruction of astrocyte distribution in the SNFL,shown in Scheme 4.

Prelaminar region

Anterior prelaminar region. Thick bodied astrocytes

IMMUNOHISTOCHEMICALSTUDYOF HUMANOPTIC NERVEHEAD ASTROGLIA 2021

SCHEME 4. Three-dimensional drawing of the SNFL. Elschning’slimiting membrane is folded back. It is made up of a very compactlayer of elongated astrocytes whose parallel processes separate thevitreous humourfrom the nerve. The elongatedastroeytes lyingbelow

this membranebecome stellate towards the PR.

separatethe ON from the surroundingretinal tissuein theanterior PR and constitute Kuhnt’s intermediary tissue[Fig. l(A)]. These cells lie in 4-5 layers that are parallelto one another and closely packed, giving them anelongated shape. The cells of the innermost layer arestellate and emit processes towards the interior of ONtissue.

The rest of the astrocytesthat make up this region arethin bodied and take two forms:elongateor stellate.Thinbodied elongated astrocytes start at Kuhnt’s limitingmembrane, where they open in a fan shape and follow astraight trajectory perpendicular to the axons. Theseastrocytesaccompanythe ciliary vesselsas they enter theON from the choroid and constitute the capillary plexusof the PR. Thin bodied stellate astrocytes are thepredominant cells in this region. Their cell bodies arebasically positionedover blood vessels and emit primaryprocesses in the direction of the vessel walls to make upan astroglialnet. The vessels follow a sinuouscourseandform a capillary plexus of irregular morphology [Fig.4(A)].

On transverse section the astrocyte perikarya andprocesses are located over the blood vessels formingcompartmentsthat surroundand bundle the ganglioncellaxons. The axon bundles are separated from one anotherby processes from these astrocytes [Fig. 4(B-D)]. Thespatial position of the compartments formed by thesecells gives the glial architecture of the region theappearance of a wicker basket in this zone [Fig.4(E,F)]. The size of the compartmentsthat make up thisglialbasket decreasesfrom anteriorto posterior.They arelargest in the regions closest to the vitreous humor [Fig.

4(B)] and very graduallybecome smaller at the posteriorPR [Fig. 4(C,D)]. This may be due to the progressiveincreasein the numberof astrocytesand blood vessels, aswell as to the structuringof the optic path.

The anterior PR contains the thin bodied astrocyteswith a single process parallel to the axons together withshort radiatingprocesses.The processes from these cellspenetratebetween the axon bundles in the interior of thecompartmentswhile their cell bodies are located on theinner wall of the compartment [Fig. 2(D)].

Scheme 5 showsa three-dimensionalreconstructionofthe glial architecturein this region,giving the appearanceof a wicker basket.

Posterior prelaminar region. Thick bodied astrocytesform a glial limiting membrane in the posterior PR thatseparates the ON from the choroid. This limitingmembrane, also known as Jacoby’s membrane, is acontinuationof Kuhnt’s intermediary tissue and has thesame characteristics [Fig, l(A)].

Most of the astrocytes in this region are thick bodiedand they form the glial ‘tubes’ through which axons run[Fig.4(G,H)]. Three to four thick primary processeswitha curved trajectoryproject from each side of the cell bodygiving it the appearanceof a crescent moon. The primaryprocesses divide into secondary processes that mix withtheprocessesof othernearby astrocytesand form the wallof the astroglial tube. The cells also emit thinnerprocesses that project in the direction of the interior ofthe glial tubes [Fig. 4(H)]. The vessels are distributedover the surface of the glial tube walls, and are coveredby processesand cellularbodiesof astrocytes[Fig.4(H)].

On transverse sections the tube walls are formed bytwo or three overlappingcells that tend to group their cellbodies in specificareas [Fig. 4(H)]. The perimeter of thetube determinesthe number of cells that make it up. Theglial tubes lie in an antero-posteriordirection within theON parallel to and in contact among themselves [Fig.4(G)]. On occasion,astrocytecell bodieswithin the tubessend processesto the tube walls, and thus divide the tubeinto two tubes of smaller diameter [Fig. 4(H)]. Thesedivisions become more frequent in the direction of theLR; the numberof glial tubes increasesantero-posteriorlywith the decrease in tube diameter. In the transitionzonebetween the posterior PR and LR, the glial tubes endpointing down the orifices that lead the axon bundles tothe pores of the lamina cribrosa [Fig. 4(I)].

The distributionof astrocytesformingglial tubes in theposteriorPR is shown in Scheme 6.

Laminar region

GFAP immunoreactivity decreases markedly in thiszone [Fig. l(A)]. Astrocytes are only lining the pores ofthe lamina cribrosa [Fig. 2(A)] where, depending on thepore perimeter, three to five cells can forma single layeraround the inside of the pore.

Astrocyte morphology is similar in the LR and theposterior PR. These thick bodied cells look like crescentmoons. Their processes join those of other astrocytesliningthe laminarcanal [Figs2(A), 4(J)] and isolatingthe

2022 A. TRIVINOet al,

FIGURE4. Captionoverleaf.

.&..

IMMUNOHISTOCHEMICALSTUDYOF HUMANOPTJCNERVE HEAD ASTROGLIA 2023

SCHEME5. Three-dimensionaldrawingof the anteriorPR glioarchitecture.Stellate thin bodiedastrocytesform compartmentsthat surroundand bundle the axons making up a basket-like structure.

collagenoustissue in the lamellae from the axonbundles.The astrocytescan emit processes into the canal betweenthe axon bundles [Fig. 4(J)], and also send processestowards the blood vessels that course near the pore walls[Fig. 4(J)]. These blood vessels, which are derived fromthe ciliary system and are less numerous than in otherregions, enter from the peripheral territories and runamong the collagen lamelle. The astrocytesin the widestcanals are frequentlystar shaped because their processesrun towards the canal wall.

Scheme 7 shows a three-dimensionalreconstmctionofthe LR in which the astrocyteslining the lamina cribrosapores can be observed.

Retrolaminar region

At this level the ON is dividedinto axonalbundlesthatare separated by connective septa. These extend eitherfrom the piamaterconnectivetissue,which penetratestheON accompanying the ciliary system pial arteries, orfrom the septa of the connective tissue surrounding thecentral vessels and sheath the arterioles exiting thecentral retinal artery. The GFAP(-) oligodendrocytesappear in the RR and their presence is indicated by anincreasednumberof nuclei [Fig.3(F)]. Four to fivelayersof thick bodied astrocytes separate ON from themeningeal sheaths in a limiting glial membrane knownas Graefe’speripheral lay,er[Fig.3(B)]. The morphologyand distributionof these astrocytesis like thatobservedinthe limitingglial membranesof the posteriorand anteriorPR. Thick bodied astrocytesare also found in the rest ofthis region. These cells can be located either between orwithin axonal bundles. In the first case the astrocytemorphologyisslightly less stellate, and their cell bodiesoverlap the connective tissue walls. The processes join

those of other astrocytes to form a glial tissue that linesthe connective septum and separates it from the axons.The primaryprocessesdivideinto thinner secondaryonesthat are directedtowardsthe insideof the axonalfascicles[Fig. 3(F)]. The morphology of these intrafascicularastrocytes is more stellate. Leaving the cell body in alldirections the processes split into thinner secondaryprocesses that subdivide the axon bundles [Fig. 3(F)].These processes may be directed towards the septumwalls [Fig. 3(F)], towards the capillaries [Fig. 3(F)] oreven towards an oligodendrocyte[Fig. 3(F)]. As in moreanteriorregions, all the RR blood vessels, are surroundedby astrocyteprocesses and cell bodies [Fig. 3(F)].

DISCUSSION

ON head as$rocytesare studied with anti-GFAPlabeling. Since GFAP is the main component of theintermediate filaments in the astrocyte cytoskeleton, theuse of antibodiesagainst this protein (Dahl et al., 1986)permits a more selective labeling of ON astrocytes thansilver stainings used in previous studies (Davis, 1940;Liss, 1956;Welter, 1957).

ASTROCYTEMORPHOLOGY

ON astrocyteshave been classifiedon the basis of thetwo astroglial lines obtained by in vitro culture (type-1and type-2) which are associated with the astrocytesobserved ‘in vivo’ (Raff et al., 1987;Raff, 1989;Milleretal., 1989).Astrocyteswith multiple radial processes andstellate appearance (Butt & Ransom, 1989; Miller et al.,1989) have been associated with the type-1 astrocyteswhich could be responsible for forming the subpiallimiting membrane and perivascular sheath. Type-2

FIGURE4. Distributionof anti-GFAPlabeled astrocytes in the different regions of the ON head. (A) Anterior PR. The thin bodied astrocyteslocated over the bloodvessels (V) form a net. (B,C,D)The distributionof the astrocytes gives the appearanceof compartments.The size of thecompartmentsis largest close to the vitreoushumor(B) anddecreasesin the directionof the posteriorPR(C and D). (E) The spatial distributionofthe astrocytes resembles a wicker basket in the anterior PR. (F) A thin bodiedastrocyte (>) can be seen inside the compartmentsof the anteriorPR. (G,H)Thickbodiedastrocytesin tbe posteriorPR.The cells formglial tubes (~) (G); these tubes are constitutedby the overlappingof severalastrocyte layers (H); the vessels (V) run along the tube walls. (I) Transition zone between the posterior PR and LR. The glial tubes (~) endpointingat the laminar pores [(>): thick bodiedastrocyte in the LR]; (J) LR:Thickbodiedastrocytes(>) onlycover the interiorperimeter of thelaminarcanal; the astrocytessendprocessesto the center of the card andto the bloodvessel (V). [CT:amective tissue; (A,F,G,I,J):longitudinalsections 30 pm; (E): longitudinalsection 6 ym; (B,C,D,H):transverse sections, 6 pm; (A,E,F,G,I,J):PAP; (B,C,D,H):PAP contrast stain with

hematoxylin;(I,J): Nomarskioptic; (A: 312x; @,C,F,G,J): 400x; (D,H,I): 500x; (E): 63x.]

2024 A. TRIVINOet al.

astrocytes could be the cells with processes orientedparallel to the nerve fibers (Butt& Ransom, 1989;Milleret al., 1989)and couldbe involvedin the formationof thenodes of Ranvier (Hidelbrand & Waxman, 1984;Ffrench-Constantet al., 1986;Black et al., 1989;Milleret al., 1989).

In this study we find two groups of astrocytes withdifferent characteristics and location: thick and thinbodied astrocytes. Our observations show that themorphologyof both groups can be stellate or elongated.We were unable to establish a correlation\between thetype-1 and type-2 astrocytes and the astrocytes weobserved in the human ON; the human cells were notso strictly specialized and could contact vessels and/oraxons as well as constitutethe glial limitingmembranes.This agrees with the observations by Suarez and Raff(1989) who demonstrated that pial and perivascularastrocytesalso emit processestowards the Ranviernodesin electron microscope observations. Therefore, theseobservations, like those by Butt and Ransom (1993), donot support the concept of astrocyte subclasses specia-lized to provideeither perinodalprojectionsor form gliallimiting membranes. These morphologicalvariations of

SCHEME6. Three-dimensionaldrawingof three astroglialtubes in theposterior PR. The thick bodied astrocytes take the form of crescentmoon and make up the wall of the tubes throughwhich the axonsrun.The vessels take a sinuous course over the surface of the walls,sometimes being covered by a process or even supporting an

occasional cell body.

elongatedor stellatehavebeen observedin both astrocytegroups and seem to be the result of their processesadapting to the surrounding structures (Chan-Ling &Stone, 1991; Hollander et al., 1991; Ramirez et al.,1994).

Our classificationof the ON astrocyteshas been madeon the basis of the amount of intermediate filament intheir cytoplasmwhich is rabeled with anti-GFAP. Thesevariations in intermediate filaments content may havefunctional implications. Thick bodied astrocyte cyto-plasmis very rich in GFAP filaments,which are supposedto confer tensile strength (Elkington et al., 1990) andthereby an important structural role in isolating andprotecting the axons from the other ON structures. Thissuppositionis further supported by the position of thesecells in the ON. They make up the limiting glialmembranes, which isolate the nerve from the retina,choroid, and meningeal sheathes; they make up theposterior PR glial tubes, which lead the axons to thelamina cribrosapores; and in the LR and RR they isolatethe axons from the connective tissue. However, thinbodied astrocytes have less GFAP filaments, indicatingtheir lower tensile strength. Located in the SNFL andanterior PR, their function may be to organize the axonsthat enter the ON. Their morphologicalfeatures indicatethey are similar to the astrocytes of the human retina(Ramfrez et al., 1994).

GLIOARCHITECTUREIN DIFFERENTON HEADREGIONS

Astrocytes in PR and LR

The density of the labeling divides the PR in twosubregions;and there is less GFAP+ tissue in the anteriorPR, which is surrounded by retinal tissue, than in theposterior PR, which is surrounded by choroidal tissue.The posteriorPR hasbeen considereda part of the laminacribrosaand called the anterior,glial, or choroidalpart ofthe lamina (Welter, 1957;Anderson et aZ.,1967;Radius,1987; Elkington et al., 1990). Other authors have evenincluded it as part of the lamina cribrosa withoutdifferentiating it (Skoff et al., 1986; Hernandez et al.,1994). Like Hogan et al. (1971), Hayreh (1978), andRamirez & Trivifio (1989), we consider that the ONregion surroundedby the choroid is part of the PR.

The LR is the only zone of the nerve in which scleralcollagenous tissue penetrates the nerve and there itconstitutesthe cribriformplates. We believe this divisionbetter agreeswith the histologicalcharacteristicsof theseregions since the PR is made up of astrocytesand axons,while the LR containsastrocytesand axons in additiontothe collagenoustissue that makes up the cribrose pores.The amount of labeled tissue decreases reflecting adecrease in the.numberof astrocytes,which only line thepores of the lamina cribrosa (Elkingtonet al., 1990).

Astrocytes in anterior PR

In this region the astrocytes are organized into abasket-like structure through whose compartments theaxons pass (Scheme 5). The astroglial nature of this

IMMUNOHISTOCHEMICALSTUDYOF HUMANOPTIC NERVE HEAD ASTROGLIA 2025

SCHEME7. Drawingof the positionof the astrocytesthat cover the innerwall of the Iaminacribrosapores. These astrocytesaresimilar to those formingthe posterior PR glial tubes; the thin bodiedastrocyteswith a long process parallel to the axons in thenerve fiber bundles are shown lying on the interior of these pores. The sinuous course of the blood vessels runs through the

cribriform plates.

basket has been demonstrated with silver staining(Welter, 1957; Hayreh & Vrabec, 1966; Anderson etal., 1967),electron microscopy(Anderson, 1969;Hoganet al., 1971)and immunohistochemistry(Elkingtonet al.,1990).

The spatial layout of the compartmentin the astroglialbasket coincides with the distribution of the vesselsfeeding this region. The vessels enter the ON from theprechoriocapillaryperipapillary choroid (Hayreh, 1978,1994; Ramirez et al., 1984; Ramirez & Triviiio, 1989)perpendicular to the axis of the ON and divide intobranches with a sinuous course that form a plexus orcapillary net. The close relation between the bloodvessels passing along the compartment walls has alsobeen observed by Welter (1957), who described theastrocytesmakingup the basketas forminga densenet ofprocesses around these capillaries.

The layout of the astrocytes at this level reflects theirsupporting and protective function with respect to theamyelinatedfibersat the place in which they bend 90 deg(Anderson, 1969; Anderson & Hoyt, 1969). Theseastrocytes may also have an important mechanical roleas they could impede possible squeezing and rubbingamong the nerve axons, since, as Welter (1957)indicated, this structure is relatively elastic if it is

comparedwith the rigidityof the scleral lamina cribrosa.This elasticity may ameliorate or prevent irreparablenerve fiber damage when the disk swells from papille-dema or neuritis.

Astrocytes in posterior PR

The thin bodied astroglial cells of the anterior aresubstitutedby thickbodied astrocytesin the posteriorPR.These cells form glial tubes throughwhich the axons run(Scheme 6). As in the anterior PR, these cells makeup astructure through which the vessels that penetrate thenerve from the adjacentchoroidrun (Hayreh, 1978,1994;Ramirez et al., 1984; Ramirez & Triviiio, 1989), andform a pericapillary net with an irregular morphology.These structureswere not noticed in the classic ‘basket’descriptionmade by Welter (1957), who considered thatthe astrocytesmaking up the basket reached the edge ofthe lamina cribrosa; only Hogan et al. (1971) noted thatthe glial septa thicken close to the lamina cribrosa.

These glial tubes may have the mechanical functionofresistingthe pressuresthat originateat this levelwhen theeyes move (Levine, 1989).The tubes surround the nervefiberslike a sheath.A numberof data seem to supportthisglial function in the posterior PR: first the abundance ofGFAPproteinsuppliesthe astroglialprocesseswith some

— —

2026 A. TRMNO et al.

tensile strength (Elkington et al., 1990) and second, thepresence of desmosornes(Anderson et al., 1967; Hoganet al., 1971) and gap junctions (Cook et al., 1973;Quigley, 1977) also supports this possibility.Both typesofjunction may have an importantrole in maintainingtheastroglial net through which the axons pass, since evenunder high osmotic pressures the gap junctions remainintact (Quigley, 1977).Additionally,in this posteriorPRthe glioarchitecture organizes the axonal bundles inpreparation for their entry to the LR.

Astrocytes in LR

The thick astroglialsheath that surroundedthe nervousfibers in the posterior PR is reduced to a single layer ofthickbodied astrocytesin the LR (Scheme7). These cellsline the interior face of the cribrose pores (Anderson,1970;Minckleret al., 1976;Elkington et al., 1990).Thedecrease in the number of glial cells can be explainedbythe presence of collagen in the lamina cribrosa, whichseparates the axons into bundles, a function that wasperformed by astrocytes in the posterior PR. So, at thislevel the only thing required of the astroglialcell layer isto isolatethe nerve fibersfrom the collagenin the walls ofthe laminar pores.

Astrocytes in RR

At the level of the RR, where the axons are perfectlybundled and myelinated,the thick bodied astrocyteshelpform the ON septa, locating themselves both intra- andinterfascicularily;their processes keep the ganglion cellaxons from directly touching the connectivepial septa.

Astrocyte-ON relation

In addition to their structural and metabolic role, ONastrocytes separate and protect the ON from thesurroundingtissues, since they make up a series of gliallimiting membranes (Elschning’s limiting membrane,Kuhnt’sintermediarytissue,Jacoby’stissueand Graefe’speripheral glial mantle). All of the membranes areconstitutedby thick bodied astrocytesthat are packagedinto various layers (Scheme 4).

The main role assigned to these membranes in the PRhas been that of constituting a barrier to the passage ofmolecules between the ON and the adjacent tissues, afunction that has been most studied in Kuhnt’s inter-mediary tissue(Tso et al., 1975;Tsukahara& Yamashita,1975; Okinami et al., 1976; Flage, 1980). Tso et al.(1975) feel that this function is also supported by theclose junctions between the astrocytes in Kuhnt’sintermediary tissue and the cells in the retinal pigmentepitheliums,as well as by the adherent areas betweenastrocytes and the external limiting membrane. Theselimiting membranes might also act as cushions againstthe rubbingthat occurs duringsmall displacementsof theON during eye movement, and thus prevent damage tothe nerve fibers that enter the nerve in the peripheralareas.

Astrocyte4100d vessel relation

Centralretina artery and vein are surroundedby a’thickastroglial limiting membrane that isolates them from thenervous parenchyme. These blood vessel–astrocyteassociations are also observed in all blood vessels ofthe ON (Liss, 1956; Welter, 1957; Hayreh & Vrabec,1966;Andersonet al., 1967;Hogan et al., 1971;Bussow,1980; Skoff et al., 1980). Sturrock (1975) suggested acorrelation between the glial population and the per-centageof vascularizedarea. This astrocyte–bloodvesselrelation is detected at the level of the ON from the timewhen the blood vessels enter the ON soon after birth; atthis time the astrocytesemit projectionsto the vesselsandisolate them from the ganglioncell axons (Dixon & Eng,1981).

One of the fundamental functions of the astrocytesmakingup the limitingglial membranesaroundthe bloodvessels is to inducethe blood-barrier(blood-brain barrierand blood+ptic nerve barrier) in the endothelial cells(Janzer, 1987; Lobrinus et al., 1992). The astrocytessecrete soluble factors (Lobrinus et al., 1992; Janzer etal., 1993) that induce unique properties including tightjunctions and molecular transport polarity in theendothelialcells (Tout et al., 1993).

RELEVANCETO GLAUCOMA

Knowledge of LR glioarchitecture is of particularinterest since experimental and histological evidenceindicatethat the damagingeffects of elevated intraocularpressure (IOP) in glaucoma affect this region first(Hayreh, 1978, 1994; Fechtner & Weinreb, 1994). Thehypothesison the causesof the changes in the head of theON caused by glaucoma include, in addition to ischemiclesionsdue to the decrease in vascularperfusion(Radius,1987; Hayreh, 1994), the mechanical alterations pro-ducedby tissue squeezing(Quigley, 1985;Radius, 1987;Minckler, 1993), which could block axonal transportand damage the axons (Quigley et al., 1981; Radius &Bade, 1982; Quigley, 1985; Minckler, 1993); astrocytesmay be more or less actively implicatedin these changes.

Thus, the first level of ON tissue affectationwould beexpectedin the astroglialcells (Goldmann,1959;Shaffer,1969) ince they are the intermediaries between thevascular system and the neurons (Kimelberg & Ransom,1986) and the small astroglial processes that penetratebetween the nerve bundles in the lamina cribrosa wouldbe involved in the metabolic support of the nerve fibersand in controlling the extracellular space (Elkington etal., 1990), as has been suggested in other parts of thecentral nervous system (Kimelberg & Ransom, 1986).Additionally,glaucoma remodels extracellular matrix ofthe lamina cribrosa (Fukuchi et al., 1992) and there ismore of the rigid Type I collagen present in glaucomathan in normal eyes (Hemandez et al., 1990;Hemandez,1993). Glaucoma also induces glial proliferation in thelamina cribrosa, apparently also demonstrated by theincrease in type IV collagen,which constitutesthe bloodvesselsand astrocytebasal membranes(Hemandez et al.,

.?IMMUNOHISTOCHEMICALSTUDYOF HUMANOPTIC NERVEHEAD ASTROGLIA 2027

1990; Morrison et al., 1990; Fukuchi et al., 1992). Theastrocyte-synthesizedextracellularmatrix can artificiallyelevateintra-axonalpressure and result in the breakdownof nornd nerve function (Quigley & Green, 1979).

REFERENCES

Abbott, N. J., Revest, P. A. & Romero, L A. (1992). Astrocyte–endothelial interaction: physiologyand pathology.Neuropathologyand Applied Neurobio@, 18, 424433.

Anderson,D. R. (1%9). Ukm.structureof human and monkey laminacribosa and optic nerve head. Archives of Ophthalmology, 82,800-814.

Anderson, D. R. (1970). Ukrastructure of the optic nerve head.Archives of O@thubno IOgy, 83, 63-73.

Anderson, D. R. & Hoyt, W. F. (1969). Ultrastructure of intraorbitalportion of human and monkey optic nerve. Archives ofOphthalmology,82, 506-530.

Anderson,D. R., Hoyt,W. F. & Hogan,M. J. (1%7). The finestructureof the astroglia in the human optic nerve and optic nerve head.Transactions of the American Ophthalmological Socie~, 65,274-305.

Black, J. A., Waxman, S. G., FriedmanB., Elmer, L. W. & AngelidesK. J. (1989).SO&urnchannels in astrocytesof rat optic nerve in situ:imrmrno-ekctronmicroscopicstudies. Glia, 2, 353–369.

BUSSOW,H. (1980).The astmcytes in the retina and opticnerve headofmammals: A spatial glia for the ganglion cell axons. Cell TissueResearch, 206, 367-378.

Butt, A. M. & Ransom, B. R. (1989). Visualization of oligodendro-cytes and astrocytes in the intact rat optic nerve by intracellularinjection of Iucifer yellow and horseradish peroxidase. Glia, 2,470-475.

Butt, A. M. & Ransom, B. R. (1993). Morphologyof astrocytes andoligodendrocitesduring development in the intact rat optic nerve.Journal of Comparative Neurology, 338, 141–158.

Chan-Ling,T. & Stone,J. (1991).Factorsdeterminingthe morphologyand distribution of astrocytes in the cat retina: a ‘contact-spacing’modeI of astrocyte interaction.Journal of Comparative Neurology,303,387-399.

Cook, R. D., Raine, C. S. & Wisnieswski, H. M. (1973). Onperivascrrlarastrocytic membrane specializations in monkey opticnerve. Brain Research, 57, 491497.

Dahl, D., Bjorklund, H. & Bignami, A. (1986). Immunologicalmarkers in astrocytes. In Federoff, S. & Vemadakis, A. (Eds),Astrocytes; Vol. III: Cell biology and patholo~ of astrocytes (pp.1-19). London:Academic Press.

Davis, F. A. (1940). GliaI cells of the normal optic nerve.Archives ofOphthalmology, 23, 735-821; 957–1018.

Dixon, R. G. & Eng, L. F. (1981).Glial fibrillaryacidic protein in theopticnerveof the developingalbinorat: an immunoperoxidasestudyof paraffin-embeddedtissue. Journal of Comparative Neurology,201, 15-24.

Elkington,A. R., Inman, C. B., Steart, P. V. & Weller R. O. (1990).The structure of the lamina cribosa of the human eye: animmunocytochemical and electron microscopical study. Eye, 4,42–57.

Fechtner, R. D. & Weinreb, R. N. (1994). Mechanismsof optic nervedamage in primaryopen angle glaucoma.Surveyof Ophthalmology,39,23-42.

Ffrench-Constant,C., Miller, R. H., Kmse, J., Schachner,M. & Raff,M. C. (1986). Molecular specialization of astrocyte processes atnodes of Ranvier in rat optic nerve. Journal of Cell Biology, 102,844-852.

Flage, T. (1980).A defect in the blood-retina barrier in the optic nervehead region in the rabbit and the monkey.Acta OphthalmoZogica,58,645-651.

Flammer, J. (1994). The vascular concept of glaucoma. Survey ofOphthalmology, 38 (supl.): S3-S6

Fukuchi, T., Sawaguchi, S., Hara, H., Shirakashi, M. & Iwata, K.(1992): ExtraCellularmatrix changes of the optic nerve lamina

cribosa in monkey eyes with experimentally chronic gkwcoma.Graefe’s Archives for Clinical and Experimemid Qrhdrafmofogy,230,421427.

Goldmann, H. (1959). Some basic problems of siarqde gia~American JournalofOphtha&rrofagy,48(Pt.11),213-220.

Hayreh,S. S. (1978). Structureandblood supplyof the optic aetve. InHeilmann,K. & Richardson,K. T. (Ed+ Glaucoma: Carrceptionsofa disease. Pathogenesis, diagnosis, therapy (pp. 78-%). Stuttgart:Georg Thieme.

Hayreh, S. S. (1994). Progress in the understandingof the vaacxdaretiology of giaucoma. Current Opim”onin OphthalmO@y, 5 ([[),26-35.

Hayreh, S. S. & Vrabec, Fr. (1966). The structure of the head of tkeopticnerve in Rhesusmonkey .American Journal of OpJrihafmology,61, 136-150.

Hemandez, M. R. (1993). ExtraceMdar matrix of the human opticnewe head: Age-related and giaucomatous changes. in Liitjesr-DrecolI, E. & Rohen, J. W. (Eds), Basic aspects of gfaucomaresearch HI (pp. 179–189). Stuttgart: Schattauer.

Hemandez, M. R., Andrzejewska, W. & Neufeld, A. H. (1990).Changesin the extracelIuIarmatrixof the humanoptic nerve.kad inprimaryopen-angleglaucoma. Anrerican JournalofOphtkako@y,109, 180-188.

Hemandez, M. R., Ye, H. & Roy, S. (1994). collagen type IV geneexpression in human optic nerve heads with primary open angleglaucoma.Experimental Eye Research, 59,41-52.

HideIbrand,C. & Waxman, S. G. (1984). Postnatal differentiation ofrat optic nerve fibers: electron microscopic observations on thedevelopmentof nodes of Ranvier and axoglial relations.JournaJ ofComparative Neurology, 224, 25-37.

Hogan,M. J., Alvarado,J. A. & Weddell, J. E. (1971).Histology of thehuman eye. An atlas and textbook (pp. 523-606). Toronto: W. B.Saunders.

Hollander,H., Makarov,F., Dreher,Z., van Driel, D., Chan-Ling,T. L.& Stone,J. (1991). Structureof the macroglia of the retina: sharingand divisionof Iabourbetween astrocytes and Muller cells. Journalof Comparative Neurology, 313, 587-603.

Janzer, R. C., Lobrinus, J. A., Darekar, P., Juillerat, L. (1993).Astrocytes secrete a factor inducirrgthe expression of HT7-proteinand neurothelin in endothelial cells of chorioallantoic vessels.Advances in Experimental Medicine and Biology, 331, 217–221.

Janzer, R. C., Raff, M. C. (1987): Aatrocytes induce blood-brainbarrier properties in endothelial cells. Nature, 325,253-257.

Kimelberg, H. K. & Ransom, B. R. (1986). Physiological andpathological aspects of astrocytic swelling. In Fedoroff, S. &Vemadakis, A. (Eds), Astrocytes, Vol. III: Cell biology andpathology of astrocytes (pp. 129–166).Orlando:Academic Press.

Levine,R. L. (1989).Organizationof astrocytes in the visual pathwaysof the goldfish: an immunohistochemical study. Journal ofComparative Neurology, 285, 231–245.

Liss, L. (1956). The astroglia of the human optic nerve, chiasma andtract. A study with silver-carbonate. Journal of Neurology, 105,151-160.

Lobrinus,J. A., Juillerat-Jeanneret,L., Darekar, P., Schlosshaver,B. &Janzer, R. C. (1992). Inductionof the blood–brainspecific HT7 andneurothelinepitopes in endothelialcells of the chick chorioallantoicvessels by a soluble factor derived from astrocytes.DevelopmentalBrain Research, 70, 207–211.

Miller, R. H., Ffrench-Constant, C. & Raff, M. C. (1989)..Themacroglial cells of the rat optic nerve. Annual Review ofNeuroscience, 12, 517–534.

Miller, R. H., Fulton,B. P. & Raff, M. C. (1989).A novel type of glialcell associated with nodes of Ranvier in rat optic nerve. EuropeanJournal of Neuroscience, 1, 172–180.

Minckler, D. S. (1993).Neuronal damage in glaucoma. In Varma, R.,Spaeth, G. L. & Parker, K. W. (Eds), The optic nerve in glaucoma(pp. 51-59). Philadelphia:J. B. Lippincott.

Minckler,D. S., McLean,L W. & Tso, M. O. M. (1976).Distributionof axonrdand glial elements in the Rhesus optic nerve head studiedby electron microscopy.American Journal of Ophthalmology, 82,179–187.

2028 A. TRIVINOet al.

Morrison,J. C., Dorman-Pease,M. E., Dunkelberger,G. R. & Quigley,H. A. (1990).Optic nerve head extracellularmatrix in primaryopticatrophy and experimental glaucoma. Archives of OphthaZmoZogy,108, 1020-1024.

Okinami,S., Ohkuma,M. & Tsukahara,T. (1976).Kuhntintermediarytissue as a barrier between the optic nerve and retina. Graefe’sArchives for Clinical and Experimental Ophthalmology, 201,57-67.

Quigley, H. A. (1977). Gap junctions between optic nerve headastrocytes. Investigative Ophthalmology and Visual Science, 16,582-585.

Quigley, H. A. (1985). The pathogenesis of optic nerve damage inglaucoma.In Beckman,H., Campbell,D. G., L’Esperance,F. A. Jr.,Mainster, M. A., Quigley, H. A., Shaffer, R. N., Simmons, R. J.,Watzke, R. C. & Wise, J. (Eds), Symposium on the laser inophthalmology and glaucoma update (pp. 111–128).St Louis: C. V.Mosby.

Quigley, H. A., Addicks, E. M., Green, W. R. & Maumenee, A. E.(1981). Optic nerve damage in human glaucoma. II. The site ofinjury and susceptibilityto damage.Archives of Ophthalmology, 99,635-649.

Quigley, H. A. & Green, W. R. (1979). The histology of humanglaucoma cupping and optic nerve damage: clinicopathologiccorrelation in 21 eyes. Transactions of American Academy ofOphthalmology and Otolaryngology, 10, 1803-1830.

Quitschke,W., Sibony,P. & Schechter,N. (1985).Variableexpressionof intermediate filamentproteins duringembryonicdevelopmentofhumanoptic nerve. Experimental Neurology, 90, 204-214.

Radius, R. L. (1987). Anatomy of the optic nerve head andglaucomatous optic neuropathy. Survey of Ophthalmology, 32,35-44.

Radius, R. L. & Bade, B. (1982). Axonal transport interruption andanatomy at the Iamina cribosa. Archives of Ophthalmology, 100,1661–1664.

Raff, M. C. (1989). Glial cell diversification in the rat optic nerve.Science, 243, 1450-1455.

Raff, M. C., Temple, S. & Ffrench-Constant, C. (1987). Glial celldevelopmentand function in the rat optic nerve. Progress in BrainResearch, 71, 435-438.

Ramfrez, J. M. & Trivirlo, A. (1989). Anatomotisiologiadel nervio6ptico. In G6mez-Ulla,F., Marfn F., Ramirez, J. M. & Triviiio,A.(Eds),La circulaci6n coroidea (pp. 31-40). Barcelona:Edika-Med.

Ramirez,J. M., Trivirio,A. & Garcia-S5nchez,J. (1984).Vasculariza-ci6rr de la cabeza del nervio 6ptico en el hombre.Archives de laSociedad Espariola de Ofialmologia, 46,413426.

Ramirez, J. M., Trivirio,A., Ramirez, A. L, Salazar, J. J. & Garcia-Stichez, J. (1994). Immunohistochemicalstudy of human retinalastroglia. Vision Research, 34, 1935-1946.

Rhodes,R. H. (1982).Developmentof the optic nerve. In Jakobiec, F.A. (J3ds), Ocular anatomy, embryolo~ and teratology (pp.601-638). Philadelphia:Harper and Row.

Shaffer, R. N. (1969).The role of the astroglial cells in glaucomatousdisc cupping.Documenta Ophthalmologic, 26,516-525.

Skoff, R. P. (1990). Gliogenesis in rat optic nerve: astrocytes aregeneratedin a single wave before oligodendrocytes.DevelopmentalBiology, 139, 149-168.

Skoff,R. P., Knapp,P. E. & Bartlett, W. P. (1986).Aatrocyticdiversityin the optic nerve: a cytoarchitectural study. In Fedoroff, S. &Vemadakis,A. (Eds), Astrocytes. Vol. I: Development, morphology,and regional specialization of astrocytes (pp. 269–291). Orlando:Academic Press.

Skoff,R. P., Toland,D. & Nast, E. (1980).Pattern of myelinationanddistributionof neuroglialcells along the developingoptic system ofthe rat and rabbit.Journal of Comparative Neurology, 191, 237–253.

Sturrock, R. R. (1975). A light and electron microscopic study ofproliferationand maturation of fibrousastrocytes in the optic nerveof the human embryon.Journal of Anatomy, 119, 223-234.

Swirez, I. & Raff, M. C. (1989). Subpial and perivascular astrocytesassociated with nodes of Ranvier in the rat optic nerve. Journal ofNeurocytology, 18, 577–582.

Tout, S., Chan-Ling,T., Hollander,H. & Stone J. (1993). The role ofMiiller cells in the formation of the blood–retinal barrier.Neuroscience, 55, 291–301.

Tso, M. O. M., Shih, C. Y. & McLean, M. L W. (1975). Is there ablood–brain barrier at the optic nerve head? Archives ofOphthalmology, 93,815-825.

Tsukahara,I. & Yamashita, H. (1975).An electron microscopicstudyof the blood+ptic nerve and fluid-optic nerve barrier. Graefe’sArchive for Clinical and Experimental Ophthalmology, 196,239-246.

Welter, J. R. (1957).The humanoptic papilla.A demonstrationof newanatomic and pathologic findings. American Journal ofOphthalmology, 44 (Pt. II), 48-64.

Acknowledgements-We thank C. F. Warren for her linguisticassistance. This work was supported by Ministerio de Educaci&r yCiencia, Direcci6n General de hrvestigaci6n Cientifica y T6cnica(DGICYT)grant PB92-0185.