Pergamon

Structural Specializations of Human Retinal GlialCellsJOSE M. RAMIREZ,*t ALBERTO TRIVINO,* ANA I . RAMIREZ,* JUAN J . SALAZAR,*JULIAN GARCIA-SANCHEZ*

Received 3 April 1995; in revised form 19 October 1995; in final form 8 November 1995

Electron microscopy and immunohistochemistry have been used to study the structuralspecializations of astrocyte and Muller glia cells in human retinas. The astrocytes and Mullercells contribute to the formation of the internal limiting membrane, the retina, the blood vessel gliallimiting membranes and the glial sheaths of the ganglion cells. Two types of junctions wereobserved among retinal glial cells . Adherent junctions were found between astrocytes and Mullercells, and between adjacent astrocytes. Gap junctions were only observed between astrocyteprocesses. These similarities suggest that astrocytes and Muller cells can perform the samefunctions in human retinas. Finally, the "perivascular astrocyte of Wolter", related only to theblood vessels, was not found . All the retinal astrocytes have the same ultrastructural characteristics,confirming the absence of these astroglial cells in human retinas observed by immunohistochemicaltechniques. Copyright © 1996 Elsevier Science Ltd.

Immunohistochemistry

There are two main types of macroglial cell in themammalian retina : Muller glia cells and astrocytes . Bothcell types are important for retinal function and bothperform a large variety of indispensable activities forneuronal survival . They maintain ionic equilibrium,regulate neuronal metabolism and participate in theblood-retina barrier as well as being involved in someretinal pathologies (Orkand et al., 1973; Laqua &Marchemer, 1975 ; Kumpulainen, 1980 ; Lam & Holly-field, 1980; Newman, 1986; Karwoski et al., 1989; Ohira& de Juan, 1990 ; Tout et al., 1993) .

Differences in morphology, orientation and cytoskele-ton composition (Bignami & Dahl, 1979; Dixon & Eng,1981) between the two cell types suggest specializedfunctions. However, ultrastructural observations in thecat retina show that both cell types contribute in the sameway to the formation of the internal limiting membrane(ILM) and blood vessel glial limiting membranes as wellas the neuronal glial sheaths (Hollander et al., 1991).

In the retina of primates, especially of man, themacroglial cells have been described in less detail than inother animal species . This is because most research hasfocused on their ultrastructural characteristics without

*Instituto de Investigaciones Oftalmologicas "Ramon Castroviejo",Facultad de Medicina, Pab . VI, 44 Planta, Universidad Complu-tense, 28.040 Madrid, Spain .

fTo whom all correspondence should be addressed [Email r amir-ezs@med.ucm.es] .

Vision Res., Vol . 36, No . 14, pp . 2029-2036, 1996Copyright © 1996 Elsevier Science Ltd . All rights reserved

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devoting any great attention to how these cells relate tothe retinal vasculature and neuronal structures (Ikui et al.,1976). It is important to understand these relationships,since astrocytes and Muller glia cells act as intermedi-aries between blood vessels and neurons, and hence anydisturbance of these cells may trigger pathologicalprocesses (Wolter, 1955a; Ohira & de Juan, 1990 ;Chan-Ling & Stone, 1992) .This paper therefore examines the ultrastructural

specializations of astrocytes and Muller glia cells in thehuman retina in order to determine their participation inthe different glial limiting membranes .

METHODS

Seven normal adult human eyes (age range : 30-60 yr),enucleated about 2-4 hr post-mortem for corneal trans-plantation were obtained from the Spanish Eye Bank forthis study. Three eyes were used for electron microscopyand the remaining four for immunohistochemical analy-sis .

Electron microscopy

Three retinas were fixed in glutaraldehyde and 0 .1 Mphosphate buffer at pH 7 .4 for 5 hr. They were thenwashed in the same buffer for a further 12 hr before beingpostfixed for 5 hr in 1% osmium tetroxide .

Tissues were dehydrated and embedded in Araldite .Sections were double-stained with uranyl acetate andlead citrate for electron microscopy study . Thick sectionswere stained for light microscopy with toluidine blue inborax .

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Astroglia Retina Human Electron microscopy

INTRODUCTION

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FIGURE 1. Electron micrographs of the internal nuclear layer . Thesoma of a Muller cell can be identified by its nucleus (N) and darkcytoplasm (arrowhead) . An astroglial light process with intermediatefilaments (arrow) can be observed next to the Muller cell (30,000x).

ImmunohistochemistryTo study astrocyte distribution the peroxidase anti-

peroxidase method was used .Four eyes were fixed with 4% paraformaldehyde in

0.1 M phosphate-buffered saline (PBS), pH 7 .4, for 4 hrat 4 °C. Two eyes were processed as retinal whole-mountsand two eyes were sectioned horizontally. The immuno-histochemical protocol followed Ramirez et ai . (1994). Amonoclonal mouse antibody directed against glialfibrillary acidic protein (GFAP clone GA-5) (Biomakor,Israel) was used in a 1/250 dilution . Retinal sections were

FIGURE 2 . Human retina stained with toluidine blue . Astrocyte nucleiare observed principally in the nerve fibre layer (NFL) and the ganglioncell layer (GCL) and may be round (arrowhead) or elongated (blackarrow) . Muller cell nuclei are angular (white arrow) and are located inthe internal nuclear layer (INL) . The Muller cells make up the internallimiting membrane (ILM) and external limiting membrane (ELM) .[External nuclear layer (ENL), external plexiform layer (EPL), internal

plexiform layer (IPL) ; 125x] .

J . M. RAMIREZ et al.

1

xFIGURE 3. Retinal astrocyte in the GCL . Nucleus (N) with abundantchromatin and heterochromatin . The cytoplasm contains : rough-surfaced endoplasmic reticulum (RER) (arrow), mitochondria (m),free ribosomes (solid arrow head), glial filaments (F) and lipofuscin (Q)

(50,000x) .

counterstained with hematoxylin for 5 min prior todehydration, clearing and mounting .

RESULTS

There are two macroglial cell types in the humanretina: Muller glial cells, which are very electron-dense(Fig. 1, arrowhead) and astrocytes, which are lesselectron-dense (Fig . 1, arrow) .

In Muller glial cells the nucleus is angular, as can beseen in the semi-thin sections with toluidine blue contrastunder light microscopy (Fig. 2; white arrow). Ultra-structurally, the cytoplasm is dark due to the presence ofmany parallel filaments (Fig . 1, arrow) . These cells lieradial to the retina and make up the two retinal limitingmembranes, the internal and the external limitingmembranes, ILM and ELM, respectively (Fig. 2), ascan be seen in the vertical sections under lightmicroscopy . The nuclei of these cells are located in theinner nuclear layer (INL) (Fig . 2) .

Ultrastructurally, astrocytes have a relatively darknucleus with abundant chromatin and heterochromatinaccumulations (Fig . 3) . Their cytoplasm contains differ-ent cytoplasmatic organelles, including : rough endoplas-matic reticulum, free ribosomes, mitochondria, Golgiapparatus, glycogen particles and dense packs ofintermediate glial filaments (Fig . 3). In older people,

STRUCTURAL SPECIALIZATIONS OF HUMAN RETINAL GLIAL CELLS

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In vertical sections (Fig. 2), astrocyte nuclei are mainlyfound in the nerve fibre layer (NFL) and ganglion celllayer (GCL) and are either round (arrowhead) orelongated (black arrow). Astrocytes are seen to take partin the formation of the ILM, which consists ofinterdigitating Miller cell endfeet and the basal mem-brane which separates the retina from the vitreous [Fig .4(a)] . Figure 4(b) shows the light astroglial processes(arrow) intercalated with the inner endfeet of Miller cells(M). Astroglial processes are less abundant than Millercell processes.

Astrocytes also participate in the formation of neuronaland axonal sheaths. Immunohistochemical preparationswith anti-GFAP showed that the elongated astrocytes andtheir processes join to form process bundles that liebetween the axonal bundles in the NFL [Fig. 5(A)] . Inhorizontal sections marked with anti-GFAP and counter-stained with hematoxylin, these astrocytes were found tomake contact with the radial peripapillar capillarieslocated in this layer [Fig . 5(B)].

Electron microscopy (Fig. 6) confirmed the location ofthe astrocyte somata (A) between the axons (ax) in theNFL, and their elongated morphology. The processesfrom these cells lie between the axons, separating theminto bundles. Where the astrocyte soma lies close to avessel (V), the astrocyte sends out processes to enfold it(arrow) . In this layer, the axonal bundles are alsosheathed by Muller cell processes (arrowhead) .Anti-GFAP labelled light microscope preparations

showed that the processes from the star-shaped astrocytesin the GCL form a honeycombed plexus around andbetween blood vessels [Fig . 7(A)] (Ramirez et al., 1994) .In horizontal sections the bodies of the ganglion cellswere found to lie within the honeycomb chambersformed by astroglial processes [Fig . 7(b)] . Electronmicroscopy showed that although the ganglion cells lieside by side they are still separated by glial processes thatessentially originate in Muller cells and form a fairlystraight continuous line between the neurons [Fig . 4(a),arrow; Fig. 8(a), black arrow] . Although occasionalastrocyte processes can be seen among these neurons,they do not form a continuous sheath, as can be seen inFig. 8(b) (white arrow) . Electron microscopy confirmedthat the astrocyte cell bodies in this layer lie close to theblood vessels, among the ganglion cells [Fig . 8(a)] .

All of the retinal blood vessels (large, intermediate andcapillaries), are surrounded by glial processes that belongto Muller cells and to astrocytes (Figs 9 and 10) . In retinalcapillaries (Fig. 9), the glial processes lie over the basalmembrane (BM). These glial processes completelyisolate the vessel from the rest of the nervous tissue,forming alternate layers of astrocyte (A) and Muller (M)cell processes . The astrocyte and Muller glia processes lievery close to the vessels . In large vessels (Fig . 10), the

astrocytes also contain dense bodies and lipofuscin . In glial cell processes lie directly on the vessel adventitia,their processes, the cytoplasmatic organelles are reduced and, as in capillaries, the Muller cell (arrow) andto a few intermediate filaments and a few mitochondria- astrocyte (arrowhead) processes form alternating layers .see Fig. 1 (arrow)-showing an astrocyte process with In this layer the astrocyte somata may lie adjacent to theonly intermediate glial filaments in the interior .

vessel wall .

FIGURE 4. Internal limiting membrane (ILM) . (a) ILM consists ofinterdigitating Muller cell endfeet (M) and the basal membrane whichaccompanies them (arrowhead). The micrograph shows how theMuller glia reaches the GCL and completely envelopes a ganglion cell,separating it from its neighbour (arrow). (GC: ganglion cell ; A:astrocyte ; ax: axons 1980x). (b) Astrocyte processes (arrow) areintercalated between Mutter glia processes (M). (BM: basal mem-

brane; ax : axon ; 5400x) .

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A

A

J. M. RAMIREZ et al.

FIG 5 B

FIG 7

FIGURE 5. Nerve fibre layer (NFL). (A) Longitudinal astroglial bundles within the NFL (arrowheads) (v, blood vessels).(Whole mount preparation, P .A .P. ;125x .) (B) Horizontal section at NFL level . The elongated astrocytes (arrows) lie parallel toone another, enveloping the capillaries (cap) with their processes . (Horizontal section . P.A.P .-hematoxylin ; 250x.)

FIGURE 7. Ganglion cell layer (GCL) . (A) Astroglial bundles with a honeycomb morphology in the ganglion cell layer . (v,blood vessels). (Whole mount preparation, P.A.P. ; 50x.) (B) The somata of the ganglion cells (GC) lie within the honeycomb

chambers (arrowheads) . (CAP: capillaries) . (Horizontal section, P .A.P .-hematoxylin; 600x.)

B

STRUCTURAL SPECIALIZATIONS OF HUMAN RETINAL GLIAL CELLS

FIGURE 6. Astrocyte (A) of the nerve fibre layer located betweenaxons (ax), with processes (arrow) enveloping a vessel (V) . The Mullercell processes can been seen among the axons (arrow head) [5400x] .

Lastly, two types of cell junction were observedbetween the glial cells : adherent junctions and gapjunctions. In Fig . 11(a) adherent junctions are appreciablebetween two adjacent astrocytes (arrowhead) or betweenMuller cells and astrocytes (arrow) . Gap junctions wereonly observed between astrocyte processes [Fig . 11(b),arrowheads] .

DISCUSSION

The ultrastructure of retinal astrocytes is similar to thatof the astrocytes in the central nervous system andpresents the same typical characteristics (Luse, 1956 ;Wendell-Smith et al., 1966) .

In agreement with observations of Hogan et al . (1971)and Ikui et al. (1976), Muller glial cells can bedistinguished from astrocytes by the marked electrondensity resulting from their abundant filamentous ma-terial and glycogen particles ; images of astrocytes arelighter because these cells have less glycogen andfilaments than the Muller cells .

Like Ikui et al. (1976), we observed no ultrastructuraldifferences between the astrocytes located close to thevessels and those located at a distance from them . In hisstudies of human retinal astrocytes using silver techni-ques, Wolter (1955a,b, 1956, 1959) described a specialtype of astroglial cell that was related only to bloodvessels, which he called a "perivascular astrocyte" .These cells were described as lying over the bloodvessels, which they surrounded with their processes,although they never contacted other structures . However,neither in an earlier paper using anti-GFAP immunohis-tochemical techniques (Ramfrez et al., 1994), nor in thepresent study using horizontal sections of the retinamarked with anti-GFAP and counterstained with hema-toxylin, were Wolter's perivascular astrocytes observedoverlying the blood vessels . All processes issued byretinal astrocytes lying over blood vessels grew away

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FIGURE 8. Ganglion cell layer . (a) The ganglion cells (GC) areseparated mainly by Muller cell processes (black arrows), forming acontinuous sheath. An astrocyte (A) lies close to the blood vessel (V)and among the ganglion cells . (White arrow : astrocyte process ; 2600x) .(b) Astrocyte processes (arrow) can be seen among the ganglion cells

(CG) but do not form a continuous sheath (21,600x) .

from the vessels . This observation was confirmed in thepresent study by electron microscopy, since all of thehuman retinal astrocytes observed here exhibit the sameultrastructural characteristics and the astrocytes locatedover the blood vessels send processes towards ganglioncells, axons or other astrocyte processes, thus confirming

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FIGURE 9. Retinal capillary in the GCL. Astrocytes (A) and Mullerglia (M) processes alternate and lie over the basal membrane (BM)

(En, endothelial cell; L, Lumen ; 21,600x).

the absence of the "perivascular astrocytes" described byWolter in human retinas .

Relations of astrocytes with different retinal structuresUltrastructural studies on the human retina, unlike

those on other mammals such as cats or monkeys, havebeen confined to analysis of the cellular characteristics ofastrocytes and Muller glia cells without delving anyfurther into the intimate relationships between these cellsand other retinal structures (Villegas, 1964; Wendell-Smith et al., 1966; Hogan et al., 1971; Ikui et al., 1976) .

FIGURE 10. Larger vessel NFL level . The glial processes (astrocytes(arrowhead) and Muller glia (arrow) lie on the vessel adventicia (AD) .The astrocyte soma (A) lie adjacent to the vessel (L, Lumen ; En,

endothelial cell ; MU, muscle cell ; 7950x) .

J . M. RAMIREZ et al.

FIGURE 11 . Cell junctions . (a) Micrograph at GCL level . Adherentjunctions are common between adjacent processes of astrocytes(arrowhead) and between the processes of an astrocyte and a Mullercell (arrow) . (GC : ganglionar cell ; 21,600x) . (b) Gap junctions areobserved between astrocyte processes (arrowheads) in the NFL

(26,400x) .

Like Hogan et al. (1971), we found that in the humanretina, astrocyte processes participate in the formation ofthe retinal ILM, along with Muller glial cells, althoughthe contribution of astrocytes is less. This has also beenobserved in other animals like monkeys (Cohen, 1961 ;Bussow, 1980) and cats (Hollander et al., 1991) .

At the level of the NFL the Muller cells and astroglialprocesses surround the axons of the ganglion cells in man(Hogan et al., 1971), monkey (Bussow, 1980) and cat(Bussow, 1980; Hollander et al., 1991). In this layer wehave further succeeded in demonstrating, both on anti-GFAP-marked horizontal sections and using electronmicroscopy, that the astrocytes make contact with the

STRUCTURAL SPECIALIZATIONS OF HUMAN RETINAL GLIAL CELLS

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radial peripapillar capillaries, thus confirming thesuggestion put forward by Ramirez et al. (1994). It isalso the case that the Muller glia cell can likewise makesuch contacts.

In the GCL, the ganglion cells in the human retina maylie adjacent to one another, but they are separated by glialprocesses belonging essentially to Muller glial cellswhich form a narrow dividing line between the neurons .This had not been described hitherto in man but had beendescribed in cat retinas by Hollander et al. (1991), whosuggested that Muller cells are specialized particularly toinsulate the ganglion cell somata in a myelin-freeenvironment. However, although we have observedastrocyte processes between neurons like Hogan et al.(1971), the processes did not form a continuous sheath .

Ikui et al. (1976) observed that in man, astroglialprocesses join together by means of desmosomes, to forman astroglial mesh parallel to the retina surface . Thismesh may serve to reinforce the capillary network andsupport the neurons present in the glial network . LikeIkui et al., we found that this astroglial mesh links thedifferent blood vessels, forming a plexus of honeycomb-like morphology . These processes not only link up viadesmosomes ; gap junctions were also observed . Desmo-somes were also found linking astrocyte and Muller gliacell processes .

This kind of junction between processes has also beenreported in other animal species . In monkeys, Bussow(1980) observed desmosomes and gap junctions betweenastrocyte processes ; in cats, Hollander et al. (1991)reported desmosomes and gap junctions between astro-glial processes together and desmosomes between Mullerglia cells and astrocytes .

Lastly, it should be noted that all the blood vessels aresurrounded by astrocyte or Muller cell processes(Villegas, 1964; Hogan et al., 1971; Ikui et al., 1976 ;Penfold et al ., 1990; Provis et al., 1991). The presentresearch showed that processes belonging to astrocytesand Muller glia cells lie in an alternating arrangement . Incapillaries, glial processes lie on the basal membrane inalternating layers of each type of glial cell, as Hollanderet al. (1991) has shown in cat retinas. However, in largervessels, the alternating glial cell layers lie on the vesseladventitia (Hogan et al., 1971 ; Penfold et al., 1990 ;Provis et al., 1991) .

Functional considerationsHollander et al. (1991) showed that the functions of

astrocytes and Muller cells are similar in cat retinas, andsuggested that they may also be similar in the humanretina. Since both cell types make up the same neuronaland axonal glial limiting membranes as well as thevascular glial sheaths, they may be variants of a singletype of macroglial cell .

It has been demonstrated physiologically that Mullercells and astrocytes perform equivalent functions . Bothcell types store glycogen and release glucose to theneurons (Ripps & Witkovsky, 1985 ; Newman, 1986).They also play an important role in regulating extra-

cellular potassium levels (Orkand et al., 1973 ; Newman,1986; Schnitzer, 1988a ; Karwoski et al., 1989). Thesecells play a decisive role in the regulation andmetabolism of neurotransmitters like GABA (Lam &Hollyfield, 1980 ; Newman, 1986; Schnitzer, 1988a ;Lopez-Colome & Romo-De-Vivar, 1991 ; Lake, 1992) .They may also help remove retinal C02, converting it tobicarbonate in a reaction catalysed by carbonic anhydrase(Kumpulainen, 1980 ; Kumpulainen et al ., 1983) .

Additionally, it has recently been proven that both celltypes (astrocytes and Muller cells) can induce blood-brain barrier properties in vascular endothelial cells (Toutet al., 1993), a function that is only performed byastrocytes in the rest of the nervous system .

If both cell types can perform the same functions, whydoes the retina have two types of macroglial cell?

There are no astrocytes in avascular retinas, but thereare Muller cells (Schnitzer, 1987). In partially vascular-ized retinas like those of the rabbit, astrocytes are onlyfound near blood vessels (Schnitzer & Karschin, 1986,1987, 1988a ; Stone & Dreher, 1987 ; Robinson & Dreher,1989; Triviiio et al ., 1992), while in totally vascularizedretinas like those of rats, cats, monkeys and humans,astrocytes are distributed over the entire retina except forthe fovea and ora serrata, the only avascular areas(Bussow, 1980; Shaw & Weber, 1983 ; Karschin et al .,1986a,b; Stone & Dreher, 1987 ; Schnitzer, 1987,1988a,b ; Distler et al., 1993 ; Ramirez et al., 1994) .

Michaelson (1954) suggested that retinal vasculariza-tion begins when the retina is thicker than 130 µm,theoretically the maximum distance that oxygen candiffuse through neural tissue, and Dreher et al . (1992)have demonstrated that Muller cells in avascular retinasare shorter than in vascularized retinas .

Moreover, according to Hollander et al. (1991), Mullercells could act as a barrier, blocking the entry ofastrocytes into avascular retinas from the optic nerve .However, astrocytes could migrate from the optic nerveinto the retina in vascularized retinas, since theappearance of vascular precursors would open spaces inthe array of the Muller cells at the level of the surface ofinner retina, and the astrocytes could move through thesespaces until they made contact with the Muller cells . Thisastrocyte distribution would only occur in zones withblood vessels ; astrocytes are not found outside these areas(Chan-Ling et al ., 1990; Chan-Ling & Stone, 1991a,b) .

All these observations suggest that vascularized retinasrequire an extra supply of macroglial cells, since there arenot enough Muller cells to perform the differentphysiological functions mentioned earlier ; thus astroglialcells may be responsible for meeting the additional needsof vascularized retinas .

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Acknowledgements-We thank Maite Solas, Agustin Fernandez andthe Centro de Microscopia Electronica (Universidad Complutense) ;and Alistair L . Ross and Carol Fox for her linguistic assistance . Thiswork was supported by DGICYT PB92-0185