Exp. Eye Res. (1992) 55. 155-162
Cell in Miiller Subretinal
Changes GARY
E. KORTE”“,
GREGORY MICHAEL
Plasma Membrane Specializations Scar Formation in the Rabbit S. HAGEMANb, MARKOd AND
DAVID V. PRATTb, STEVEN AVINOAM OPHIR”
During GLUSMAN”,
aDepartments of Ophthalmology and Anatomy, Montefiore Medical Center/Albert Einstein College of Medicine, Bronx, NY, b Bethesda Eye Institute, Department of Ophthalmology, St Louis University School of Medicine, St Louis, MO, c Department of Neurology, Oregon Health Sciences University, Portland, OR, d NIH Biological Microscopy and Image Reconstruction Resource, Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY, U.S.A. and eDepartment of Ophthalmology, Hadassah University Hospital, Jerusalem, Israel (Received
Houston
76 August
1991 and accepted
in revised form 1 November
1991)
The aim of this study was to identify changes in Miiller cell plasma membrane specializations during experimentally induced subretinal gliosis in rabbits. When rabbits are dosed with sodium iodate, large expanses of retinal pigment epithelium and photoreceptors are destroyed. They are replaced by a subretinal scar consisting mainly of the ascending processes of MiiUer cells, These processes transform from the slender, highly polarized structures seen in normal animals into irregnlar processes that form a glia liiitans along the basement membrane of the pigment epithelium, left bare following its degeneration. As the scar processes extend through the subretinal space and contract this basement membrane, they undergo dramatic changes in shape that are especially apparent in three-dimensional computer reconstructions of serial thick sections examined by high-voltage electron microscopy. Other changes involve the intercellular junctions and apical microvilli normally associated with the external limiting membrane. These structures become scattered over the surfaces of the ascending processes and are eventually lost. Loss of microvilli is associated with disappearance of immunostaining for a specific glycoconjugate normally associated with the microvillar plasma membrane. The observations document profound changes in Miiller cell structural and functional polarity during subretinal scar formation. key words : glia : glycoconjugate ; Miiller cell : plasma membrane ; rabbit ; ultrastructure.
1. Introduction Miiller cells form subretinal scars during severi pathologic and induced conditions, e.g. when laser treatments and retinal detachments cause destruction of retinal pigment epithelium or photoreceptors (Ikui, 1974; Ishiiawa, 1974; Laqua and Machemer, 1975; Erickson et al., 1983; Lewis et al., 1989). The Miiller cell response includes hypertrophy, proliferation, increased expression of glial fibrillary acidic protein and vimentin as well as their messenger RNAs, and the movement of Miiller cell somata into the subretinal space, via migration from the inner nuclear layer. The most striking structural changes that occur in Miiller cells during subretinal scar formation involve the slender ascending processes that form the external limiting membrane (ELM). As these processes move into the subretinal space, they transform into stout, club-like processes that no longer contribute to the ELM (Ikui 1974; Ishikawa, 1974; Laqua and Machemer, 1975; Erickson et al., 1983). The aim of this study was to determine if there are also changes in the plasma membrane specializations of the as* For correspondence at: Department of Ophthalmology. MontefioreMedicalCenter. 11 I E. 210th Street,Bronx, N.Y. 10467, U.S.A.
00144835/92/070155
+08 $08.00/O
tending processes, in particular the microvilli and the intercellular junctions that comprise the ELM. To study these changes, we used light and electron microscopy to examine the transformation of Miiller cell ascending processes during experimentally induced subretinal scar formation in rabbits that received intravenous injections of sodium iodate. Sodium iodate initially destroys the retinal pigment epithelium (Graymore, 19 70 ; Bernstein, Carlson and Bok, 1991). This is followed by photoreceptor de-
generation
and activation
of Miiller
cells to form a
subretinal scar. We observed striking changes in the shape of ascending processes, the placement of
intercellular
junctional
complexes and microvilli
over
their surfaces, and the loss of a microvilli-associated antigen during subretinal scar formation. These observations have been reported in preliminary fashion (Glusman and Korte, 1988: Pratt et al., 1988; Korte, 1990).
2. Materials
and Methods
Electron Microscopy Mature female, pigmented New Zealand rabbits were used. Observations were made on three normal rabbits and on ten rabbits that received intravenous 0 1992 Academic Press Limited
156
injections of sodium iodate in normal saline, as previously described (Korte, Reppucci and Henkind. 1984). One to 11 weeks after administration of sodium iodate the rabbits were euthanized by intravenous overdose of sodium pentobarbital (145 mg kg-‘) and the eyes immediately removed. The cornea, lens and vitreous were removed and the remaining eyecup immersed in 2 % formaldehyde and 2 ‘$, glutaraldehyde in 0.1 M phosphate buffer, pH 7.2. In some cases 0.5 % tannic acid was included in the fixative. After fixation overnight at 4°C slices of tissue were cut with a razor blade in the central-peripheral direction, rinsed in phosphate buffer, immersed for 2 hr in 2% osmium tetroxide in phosphate buffer, dehydrated in methanol and embedded in epoxy resin. Sections 2-3 ,um thick were stained with toluidine blue for light microscopy.
G. E. KORTE
ET AL.
Thin sections were stained with uranyl acetate and lead citrate prior to electron microscopic examination at 80 kV. For high-voltage electron microscopy (HVEM) sections 0.5 or 1 pm thick were mounted on Formvarcoated slot grids, stained with uranyl acetate and lead citrate and examined at 1000 kV at the NIH Biological Microscopy and Image Reconstruction Resource located at the New York State Department of Health in Albany, New York. Continuous series of sections were used to make computer reconstructions (Marko, Leith and Parsons, 1988). In this procedure, a digitizer is used to make tracings from stereo pairs of photographic negatives of serial sections through Miiller cells. A computer program (’ Sterecon ’ ; see Marko, Leith and Parsons, 1988) then generates stereo
FIG. 1. Light micrographs of subretinal scars. A, Site of subretinal scar formation (arrow), flanked by zones of RPE lossand photoreceptoratrophy. Scallopedzoneat left issimilarto rosettesseenin (B). Note that sitesflanking the subretinalscarstill have intact ELM, as seen in electron micrographs from comparable areas [cf. Fig. 3(A)]. Two weeks after iodate. Plastic section stained
with toluidine blue, x 120. B, Several photoreceptor rosettes have been derived from the scalloped photoreceptor layer. Four weeks after iodate. Paraffin section stained with hematoxylin and eosin. x 320. C, In formative subretinal scars the external limiting membranebreaksdown and Miiller cell processes (P) move into the subretinalspaceand approachthe RPE basement membrane (at arrowhead, necrotic RPE has been removed). Among the Miiller cell processes are mitotic cells (large arrow, probably a mitotic Miiller cell) and several other cells (small arrows). The inner nuclear layer occupies the bottom third of the picture. One week after iodate. Plastic section stained with toluidine blue. x 940. D, In a mature scar (s) cell bodies [e.g. those seen at small arrows in (C)] are infrequent, the scar being formed mainly by hypertrophic ascending Mtiller cell processes (arrow) that extend up to the RPE basement membrane. Directly beneath the scar is remnant outer nuclear layer, consisting of photoreceptor somata and pale, stubby inner segments. Miiller cell endfeet bordering the vitreous (at bottom of picture) appear hypertrophied. One month after iodate. Plastic section stained with toluidine blue. x 300.
MijLLER
CELL
STRUCTURE
reconstructions that can be examined from any desired angle. Immunohistochemistry A Miiller cell microvillar antigen labeled by a monoclonal antibody, designated 22/G9 (Pratt, Johnson and Hageman, 1987; Pratt, Hageman and Johnson, 1988), was localized by immunohistochemistry. The eyes of two rabbits were fixed in 4% formaldehyde in 01 M phosphate buffer, pH 7.2, 3 weeks after administration of sodium iodate. The eyecups were fixed for 2-3 hr, embedded in acrylamide and sections 5-6 pm thick were cut on a cryostat at -20°C (Johnson and Blanks, 1984). Non-specific binding sites were blocked with 1 mg ml-’ globulinfree bovine serum albumin in 10 mM phosphatebuffered saline containing 1 mM magnesium chloride and 1 mM calcium chloride (PBMC). Sections were rinsed briefly and then incubated for 60 min at room temperature with mouse monoclonal antibody 22/G9, diluted l/100-200 in PBMC. After rinsing in PBMC for 10 min, sections were incubated in affinity-purified, fluorescein-conjugated goat anti-mouse IgG antibody in PBMC for 30-60 min at room temperature in the dark. Slides were coverslipped with Immu-Mount (Shandon) and examined by epifluorescence microscopy. Controls included (1) incubation of sections with secondary antibody alone and (2) incubation of sections in normal mouse serum followed by secondary antibody. 3. Results The retinopathy induced by sodium iodate in pigmented rabbits is manifest initially as necrosis of the RPE, which begins as early as 6 hr after administration of sodium iodate (unpubl. obs. ; see Anstadt
157
et al., 1982). Necrosis of the pigment epithelium is fulminant within 1 day of administration of iodate and is followed several days later by atrophy of the photoreceptors (e.g. shortening of the outer segments). We report observations made from 1 week to 1 month after iodate administration, since subretinal scars in varying degrees of formation are scattered through the fundus. Light Microscopy By l-2 weeks after administration of sodium iodate, scars form where the subretinal space is vacant due to photoreceptor atrophy and removal of the RPE by macrophages [Figs l(A)-(C)]. At these sites the outer nuclear layer and ELM often appear scalloped due to formation of photoreceptor rosettes [Figs l(A) and (B)]. One month after iodate adminstration, rosettes are no longer present and numerous well-developed subretinal scars occupy the tissue space formerly occupied by the RPE and photoreceptors [Fig. l(D)]. Immunostaining for the antigen detected by monoclonal antibody 22/G9 (see Materials and Methods) was localized to the apices of Miiller cells in normal rabbits and, in rabbits that received sodium iodate, wherever the ELM was intact, i.e. in remnant normal retina and in rosettes [Figs 2(A) &d (B)]. The staining disappeared where the ELM had broken down [arrows in Fig. 2(B)]. In sections stained with toluidine blue these sites corresponded to regions where the ELM was disrupted due to photoreceptor atrophy and subretinal scar formation, e.g. the arrow in Fig. l(A) and the zone between the rosettes seen in Fig. l(B). In sections examined by transmission electron microscopy these sites corresponded to areas where the Miiller cell ascending processes had penetrated beyond the ELM and into the subretinal space [Fig. 3(A)].
FIG. 2. Immunofluorescence for Miiller cell microvillar antigen detected by monoclonal antibody 22/G9, from a rabbit that received sodium iodate 3 weeks before being killed. A, In area of normal retina the ELM stains intensely. It appears ragged due to staining of individual microvilli. x 250. B. Where the ELM is disrupted (arrows) due to subretinal scar formation, immunoreactivity
is absent. The ELM encircles the lumina
of rosettes. x 250.
158
G. E. KORTE
ET AL.
FIG. 3. Electron micrographs from thin sections through the edge of a scar, examined 2 weeks after iodate. The locale correspor Ids to the edge of the area denoted by the arrow in Fig. l(A). A, A Miiller cell ascending process (P) sends .several branches I (straight arrows) into the scar while another branch (curved arrow) still contributes to the external lir niting membrar le [detailed in (B)]. The scar processes are irregular in profile and form endfeet at the former RPE basement mem lbrane (arrowhe :ads, detailed in inset). Several photoreceptor somata (n, a nucleus) are seen within the scar. IS denotes inner seg ments of atroph :ic photoreceptors. Cell at left is probably a macrophage, as melanosomes apparently within phagosomes were s’een in the part (If the cell outside this field. x 7900. Inset, Detail of endfoot of RPE basement membrane. Plasma membrane here forms
DULLER
CELL
159
STRUCTURE
FIG. 4. Remant zonulae adherentes and microvilli along the length of scar processes, seen in a thin section (A) and a computer reconst~ction of serial thick sections ex~ed by HW (B). A, Two adjacent scar processes (a light one and a dark one-otherwise similar in their cytology) are connected by xonulae adherentes (za) of the external hmtting membrane type (note extensive cytoplasmic density and focal appositions of plasma membrane) with a few microvilii (arrow). The processes contain abundant glycogen granules and intermediate iYaments. Scars often contain Miiller cell processesof differing electron densities, perhaps related to their different content of intermediate filaments. x 40 600. B, Computer reconstruction of ascending Miiller cell process in a series of O-S-pm thick sections through a scar examined by HYEM, from a rabbit obtained 6 weeks after iodate. The process abuts the basement membrane formerly occupied by the RPE, at top of picture. Along the length of the process two clusters of microvilli [denoted as a solid light zone, since every ~cro~us cannot be traced ; the space between the tracing of the microvillar xone and the tracing of the scar process is due to inaccuracy (e.g. hand movements) during tracing of the structures] are adjacent to external limiting membrane xonulae adherentes, denoted by white bars beneath plasma membrane.
The changes that ascending Miiller cell processes undergo during scar formation were studied in thin sections where the normal ELM was contiguous with a site of scar formation. Here, normal ascending processes con~but~g to subret~al scars arose in proximity to each other, sometimes from the same cell, and could be compared [Fig. 3(A) and (B)]. The Muller cell ascending processes still contributing to ELM were slender, contained numerous ~~chondria, had apical microvilli and formed zonulae adherer&es that consti~te the ELM [Fig. 3(B)]. Those processes contributing to the scar, however, had transformed
contained
into stout, irregular
abundant
intermediate
processes
that
filaments and gly-
cogen granules, and formed endfeet with attachment sites on the remnant RPB basement membrane [Fig. 3(A) and inset]. Apparent remnants of ELM junctional
complexes and associated microvilli were observed along their length in thin sections and in reconstructions of serial thick sections examined by HVEM [Fig. 4(A) and (B)]. The shape of the scar processes varied in younger and older scars [e.g. those seen in Figs l(C) and (D). respectively]. ~mputer reconst~ctions of three ascending processes in a forming subretinal scar revealed a more irregular surface contour than in three reconstructions of ascending processes from an older scar [cf. Figs S(A) and (B)]. This correlated with the fine structure of over 50 scars examined [e.g. Fig. 3(A) from a young scar, and Fig. S(C) from an old scar]. Contact with the RPE basement membrane seemed to influence the shape of the Miiller cell ascending process. In the process illustrated in the stereo pairs in Fig. 6(A), several appendages are seen on the part of the process nearest the viewer, which is not in contact
attachment sites (arrowheads) and has an underlying meshwork of fine filaments. The cytoplasm contains abundant glycogen granules and interme~ate filaments, detailed in Fig. 4(A). From tissue mordanted with tannic acid. x 50000. B, Detail of portion of Miiller cell process seen at curved arrow in (A), from an adjacent section. The process contains numerous mitochondria and still possesses microvilli (arrows) and zonulae adherentes (za) that constitute the external limiting membrane. Scar processes [e.g. those seen in (A)] generally contain fewer mitochondria but more intermediate filaments and glycogen. x 19600.
160
G. E. KORTE
ET AL
FIG. 5. Computer reconstructions of scar processes in a set of 1-ym thick sections examined by HVEM, from a rabbii t obtained week .s after iodate. (A) is from a young, or early. scar [e.g. processes similar to those seen in Figs l(C) and 3(A)]. ( B) is from a more advanced scar [e.g. processes like those seen in Fig. l(D)]. The stereo electron micrographs in (C) are printed I from the stereo Inegatives of the first section of the series reconstructed in (B). A. This reconstruction is tilted so that the irregu liar shape of the Ejrocess is seen to advantage, e.g. the irregular, smaller process that arises off the right side of the larger one (at the black arrow) and curves downward to contribute to the external limiting membrane, its junctions indicated by the white bar s beneath 6
MijLLER
CELL
STRUCTURE
161
with the basement membrane. However. when the reconstruction is ‘ split ’ [Fig. 6(B)] to reveal the part of the process actually in contact with the basement membrane [and buried in the stack of sections of the reconstruction in Fig. 6(A)], the loss of appendages at this site becomes apparent. The somata and descending processes of Miiller cells at sites of subretinal scar formation appeared normal except for apparent hypertrophy of the foot processes at the inner limiting membrane [e.g. Fig. l(D)]. A detailed analysis of these parts of the Miiller cell, however, was not undertaken, as the most striking changes occurred in the ascending processes. 4. Discussion During
subretinal
scar formation,
the ascending
Miiller cell processes move into the subretinal space and occupy the tissue volume formerly occupied by photoreceptors, interphotoreceptor matrix and pigment epithelium.
Eventually
the processes
reach the
pigment epithelium basement membrane, where they form a glia limitans consisting of glial endfeet lie those abutting retinal blood vessels and the inner limiting membrane (cf. Fig. 11-9 of Hogan, Alvarado and Weddell, 1971). In the interim between moving from the level of the ELM to the basement membrane of the pigment epithelium, the Miiller cell plasma membrane specializations change. While new attachments to the RPE basement membrane form, the microvilli and inFIG. 6. HVEM computer reconstruction of a scar process that runs tangential to the RPE basement membrane. The orientation is 90” clockwise from the reconstructions seen in Fig. 5. A, In the stereo reconstruction the process bears many finger-like processes directed towards the RPE basement membrane (linear profiles at left). B, The same reconstruction seen in (A) is now presented as a stack profile instead of in stereo. The stack is ‘split ’ so that the series of sections through the portion of the process actually contacting the RPE basement membrane (on left) and not observed in the stereo reconstruction in (A), can be compared with the series of sections not bordering the membrane (on right) but which are seen in the reconstruction in (A). Note that the surface of the process contacting the basement membrane is smooth, while that not bordering it has numerous finger-like projections. These latter hid the flat area bordering the basement membrane in the stereo reconstruction seen in (A).
tercellular junctional complexes located at the ELM become scattered over the surface of the ascending process and are eventually lost, Coincident with ELM breakdown and loss of microvilli is the loss of a specific microvilli-associated glycoconjugate recognized by an antibody designated 22/G9 (Pratt, Johnson and Hageman, 1987; Pratt, Hageman and Johnson, 1988). The loss of reactivity for this antigen accom-
panies the loss of cytological polarity of the Miiller cell ascending process during scar formation. This loss of staining may be due to several factors, e.g. diffusion of the antigen through the plane of the plasma membrane to such an extent that it is no longer detected by the procedure used, internalization and degradation of plasma membrane, or loss of epitopes during gliosis. Clearly this phenomenon warrants further study as a
the plasma membrane. The space intervening between these junctions and those of a portion of an adjacent scar process (white arrow) was occupied by the inner segment of an atrophic photoreceptor. This was not included in the reconstruction because it would have obscured reconstruction of the glial processes. Note that the surface facing the RPE basement membrane (linear profiles at top) is flat. B, Reconstruction of a process from an older scar in the same animal as that depicted in (A) [e.g. those seen in Fig. l(D)]. Compared to the process reconstructed in (A), this one is more regular and does not contribute to the external limiting membrane. An electron micrograph of one of the thick sections used to reconstruct this process is seen in (C). C, Stereo HVEM picture of the first l-pm thick section used to make the reconstruction in (B). Note how the reconstruction from the subsequent series of sections has provided more information than is present in this single section, e.g. the extent of the two small processes that surround an adjacent photoreceptor soma (s) and how discrete endfeet are formed at the RPE basement membrane at the top of the picture. x 3500. 11
EER55
162
mechanism of remodeling the Miiller cell plasma membrane during scar formation. One advantage of the sodium iodate retinopathy model in rabbits is that the pigment epithelium does not remain intact, providing an opportunity to examine the response of the Miiller cell processes to contact with a novel basement membrane. To this extent the model tests the hypothesis that at sites where Miiller cells form a glia limitans (e.g. at the inner limiting membrane of the retina or around retinal blood vessels) a basement membrane is always present and provides cues for the formation of glial endfeet and their cytologic specializations, such as attachment sites. When brought into contact experimentally with the RPE basement membrane, Miiller cell endfeet identical to those at retinal blood vessels and the inner limiting membrane are observed. The plasma membrane in contact with basement membrane seems to respond in two ways: it forms attachment sites, and it loses the uneven contour seen in Miiller cell plasma membrane that does not appose the basement membrane. This contact seems to precede, and perhaps signal shape changes in the rest of the process, for those processes containing the basement membrane seem more uniform in contour than those that do not. Although the signal molecules involved in eliciting these changes have not been identified, it is possible that basement membrane constituents such as fibronectin or laminin are involved. The responsiveness of glia to these molecules and several growth factors that may be present during subretinal scar formation (e.g. fibroblast growth factors from macrophages) has been documented (see Harvey, Roberge and Hjelmeland, 1987). The rabbit model used in this study may be suitable for identifying those factors responsible for controlling the differentiation of the Milller cell and its ‘redifferentiation ’ during subretinal scar formation. Acknowledgements Supported by NE1 grants EY08284 (G.E.K.) and EY06463 (G.S.H.) ; unrestricted departmental grants from Research to Prevent Blindness, Inc to the Department of Ophthalmology, Albert Einstein College of Medicine and to the Bethesda Eye Institute ; and a grant from NIH (RR01 12 19) supporting the Biological Microscopy and Image Reconstruction Resource located at the New York State Department of Health in Albany, New York. References Anstadt, B., Blair, N., Rusin, M., Cunha-Vaz, J. and Tso, M. (1982). Alteration of the blood-retinal barrier by sodium iodate: kinetic vitreous fluorophotometry and
G. E. KORTE
ET AL.
horseradish peroxidase tracer studies. Exp. Eye Res. 35. 653.
Bernstein,P., Carlson.A. and Bok, D. (1991). The retinyl ester synthetase is a cellular target for the retinal pigment epithelium toxin sodium iodate. Invest. Ophthalmol.Vis. Sci. (Suppl.)32, 1250. Erickson,P., Fisher,S..Anderson,D., Stern,W. andBorgula, G. ( 1983). Retinaldetachmentin the cat: outer nuclear and outer plexiform layers.Invest. Ophthalmol. 24. 92 7. Glusman.S.,Korte, G. (1988). Miiller cellresponseand scar formation during sodiumiodateretinopathy in rabbits. Invest. Ophthalmol. Vis. Sci. (Suppl.)29, 289. Graymore, C. (1970). Biochemistry of the retina. In Biochemistryof the Eye. (Ed. Graymore. C. N.) Pp. 645-735. AcademicPress:New York. Harvey, A., Roberge. F. and Hjelmeland, L. (1987). Chemotaxisof the rat retinal glia to growth factors found in repairing wounds.Invest. Ophthalmol. Vis. Sci. 28, 1092-g. Hogan,M., Alvarado, J. and Weddell,J. (1971). Histology of the HumanEye. W. B. Saunders:Philadelphia. Ikui, H. (1974). Morphology and pathology of the retinal glia. Acta Ophthalmol. (Jap.) 75, 1245. Ishikawa, Y. (1974). Histological studies of repairing processes after xenon photocoagulationin the monkey retina. I. Electron microscopic studies on cellular responses in the early repairing stage.Acta Ophthalmol. (Jap.)78, 606. Johnson, L. V. and Blanks, J. C. (1984). Application of acrylamideasan embeddingmediumin studiesof lectin and antibody-bindingin the vertebrate retina. Curr. Eye Res. 3, 969-74.
Korte, G. E. (1990). High voltage electron microscopy of reactive Mtiller cellsin the rabbit retina. Anut. Rec. 226, 52A. Korte, G. E., Reppucci, V. and Henkind, P. (1984). RPE destruction causes choriocapillaris atrophy. Invest. Ophthalmof. Vis. Sci. 25, 1135. Laqua. H. and Machemer,R. ( 1975). Glial cell proliferation in retinal detachment (massive periretinal proliferation). Am. J. Ophthalmol. 80, 602. Lewis,G.. Erickson,P., Guerin. C., Anderson,D. and Fisher, S. (1989). Changesin the expressionof specificMiiller cell proteinsduring long term retinal detachment.Exp. Eye Res. 49, 93.
Marko, M.. Leith, A. and Parsons, D. (1988). Threedimensionalreconstructionof cellsfrom serialsections and whole-cellsmounts usingmultilevel contouring of stereo micrographs. 1. Electron Microscopy Tech. 9, 393.
Pratt, D. V., Hageman, G. S. and Johnson,L. V. (1988). Immunologicalcharacterizationof an antigenexpressed by Miiller cell apical microvilli. Invest. Ophthalmol. Vis. Sci. (Suppl.) 29, 244. Pratt. D., Johnson, L. V. and Hageman, G. S. (198 7). Polarizeddistribution of a Miiller cell-specificantigen. Invest. Ophthalmol. Vis. Sci. (Suppl.)28, 258. Pratt, D.. Korte, G. E., Johnson,L. V. and Hageman.G. S. (1988). Altered expressionof a Miiller cell-specific antigen in sodium iodate induced retinopathy. Eight International Congresson EyeResearch.Proc. Intl. Sot. Eye Res. 5, 124.
Cell in Miiller Subretinal
Changes GARY
E. KORTE”“,
GREGORY MICHAEL
Plasma Membrane Specializations Scar Formation in the Rabbit S. HAGEMANb, MARKOd AND
DAVID V. PRATTb, STEVEN AVINOAM OPHIR”
During GLUSMAN”,
aDepartments of Ophthalmology and Anatomy, Montefiore Medical Center/Albert Einstein College of Medicine, Bronx, NY, b Bethesda Eye Institute, Department of Ophthalmology, St Louis University School of Medicine, St Louis, MO, c Department of Neurology, Oregon Health Sciences University, Portland, OR, d NIH Biological Microscopy and Image Reconstruction Resource, Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY, U.S.A. and eDepartment of Ophthalmology, Hadassah University Hospital, Jerusalem, Israel (Received
Houston
76 August
1991 and accepted
in revised form 1 November
1991)
The aim of this study was to identify changes in Miiller cell plasma membrane specializations during experimentally induced subretinal gliosis in rabbits. When rabbits are dosed with sodium iodate, large expanses of retinal pigment epithelium and photoreceptors are destroyed. They are replaced by a subretinal scar consisting mainly of the ascending processes of MiiUer cells, These processes transform from the slender, highly polarized structures seen in normal animals into irregnlar processes that form a glia liiitans along the basement membrane of the pigment epithelium, left bare following its degeneration. As the scar processes extend through the subretinal space and contract this basement membrane, they undergo dramatic changes in shape that are especially apparent in three-dimensional computer reconstructions of serial thick sections examined by high-voltage electron microscopy. Other changes involve the intercellular junctions and apical microvilli normally associated with the external limiting membrane. These structures become scattered over the surfaces of the ascending processes and are eventually lost. Loss of microvilli is associated with disappearance of immunostaining for a specific glycoconjugate normally associated with the microvillar plasma membrane. The observations document profound changes in Miiller cell structural and functional polarity during subretinal scar formation. key words : glia : glycoconjugate ; Miiller cell : plasma membrane ; rabbit ; ultrastructure.
1. Introduction Miiller cells form subretinal scars during severi pathologic and induced conditions, e.g. when laser treatments and retinal detachments cause destruction of retinal pigment epithelium or photoreceptors (Ikui, 1974; Ishiiawa, 1974; Laqua and Machemer, 1975; Erickson et al., 1983; Lewis et al., 1989). The Miiller cell response includes hypertrophy, proliferation, increased expression of glial fibrillary acidic protein and vimentin as well as their messenger RNAs, and the movement of Miiller cell somata into the subretinal space, via migration from the inner nuclear layer. The most striking structural changes that occur in Miiller cells during subretinal scar formation involve the slender ascending processes that form the external limiting membrane (ELM). As these processes move into the subretinal space, they transform into stout, club-like processes that no longer contribute to the ELM (Ikui 1974; Ishikawa, 1974; Laqua and Machemer, 1975; Erickson et al., 1983). The aim of this study was to determine if there are also changes in the plasma membrane specializations of the as* For correspondence at: Department of Ophthalmology. MontefioreMedicalCenter. 11 I E. 210th Street,Bronx, N.Y. 10467, U.S.A.
00144835/92/070155
+08 $08.00/O
tending processes, in particular the microvilli and the intercellular junctions that comprise the ELM. To study these changes, we used light and electron microscopy to examine the transformation of Miiller cell ascending processes during experimentally induced subretinal scar formation in rabbits that received intravenous injections of sodium iodate. Sodium iodate initially destroys the retinal pigment epithelium (Graymore, 19 70 ; Bernstein, Carlson and Bok, 1991). This is followed by photoreceptor de-
generation
and activation
of Miiller
cells to form a
subretinal scar. We observed striking changes in the shape of ascending processes, the placement of
intercellular
junctional
complexes and microvilli
over
their surfaces, and the loss of a microvilli-associated antigen during subretinal scar formation. These observations have been reported in preliminary fashion (Glusman and Korte, 1988: Pratt et al., 1988; Korte, 1990).
2. Materials
and Methods
Electron Microscopy Mature female, pigmented New Zealand rabbits were used. Observations were made on three normal rabbits and on ten rabbits that received intravenous 0 1992 Academic Press Limited
156
injections of sodium iodate in normal saline, as previously described (Korte, Reppucci and Henkind. 1984). One to 11 weeks after administration of sodium iodate the rabbits were euthanized by intravenous overdose of sodium pentobarbital (145 mg kg-‘) and the eyes immediately removed. The cornea, lens and vitreous were removed and the remaining eyecup immersed in 2 % formaldehyde and 2 ‘$, glutaraldehyde in 0.1 M phosphate buffer, pH 7.2. In some cases 0.5 % tannic acid was included in the fixative. After fixation overnight at 4°C slices of tissue were cut with a razor blade in the central-peripheral direction, rinsed in phosphate buffer, immersed for 2 hr in 2% osmium tetroxide in phosphate buffer, dehydrated in methanol and embedded in epoxy resin. Sections 2-3 ,um thick were stained with toluidine blue for light microscopy.
G. E. KORTE
ET AL.
Thin sections were stained with uranyl acetate and lead citrate prior to electron microscopic examination at 80 kV. For high-voltage electron microscopy (HVEM) sections 0.5 or 1 pm thick were mounted on Formvarcoated slot grids, stained with uranyl acetate and lead citrate and examined at 1000 kV at the NIH Biological Microscopy and Image Reconstruction Resource located at the New York State Department of Health in Albany, New York. Continuous series of sections were used to make computer reconstructions (Marko, Leith and Parsons, 1988). In this procedure, a digitizer is used to make tracings from stereo pairs of photographic negatives of serial sections through Miiller cells. A computer program (’ Sterecon ’ ; see Marko, Leith and Parsons, 1988) then generates stereo
FIG. 1. Light micrographs of subretinal scars. A, Site of subretinal scar formation (arrow), flanked by zones of RPE lossand photoreceptoratrophy. Scallopedzoneat left issimilarto rosettesseenin (B). Note that sitesflanking the subretinalscarstill have intact ELM, as seen in electron micrographs from comparable areas [cf. Fig. 3(A)]. Two weeks after iodate. Plastic section stained
with toluidine blue, x 120. B, Several photoreceptor rosettes have been derived from the scalloped photoreceptor layer. Four weeks after iodate. Paraffin section stained with hematoxylin and eosin. x 320. C, In formative subretinal scars the external limiting membranebreaksdown and Miiller cell processes (P) move into the subretinalspaceand approachthe RPE basement membrane (at arrowhead, necrotic RPE has been removed). Among the Miiller cell processes are mitotic cells (large arrow, probably a mitotic Miiller cell) and several other cells (small arrows). The inner nuclear layer occupies the bottom third of the picture. One week after iodate. Plastic section stained with toluidine blue. x 940. D, In a mature scar (s) cell bodies [e.g. those seen at small arrows in (C)] are infrequent, the scar being formed mainly by hypertrophic ascending Mtiller cell processes (arrow) that extend up to the RPE basement membrane. Directly beneath the scar is remnant outer nuclear layer, consisting of photoreceptor somata and pale, stubby inner segments. Miiller cell endfeet bordering the vitreous (at bottom of picture) appear hypertrophied. One month after iodate. Plastic section stained with toluidine blue. x 300.
MijLLER
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reconstructions that can be examined from any desired angle. Immunohistochemistry A Miiller cell microvillar antigen labeled by a monoclonal antibody, designated 22/G9 (Pratt, Johnson and Hageman, 1987; Pratt, Hageman and Johnson, 1988), was localized by immunohistochemistry. The eyes of two rabbits were fixed in 4% formaldehyde in 01 M phosphate buffer, pH 7.2, 3 weeks after administration of sodium iodate. The eyecups were fixed for 2-3 hr, embedded in acrylamide and sections 5-6 pm thick were cut on a cryostat at -20°C (Johnson and Blanks, 1984). Non-specific binding sites were blocked with 1 mg ml-’ globulinfree bovine serum albumin in 10 mM phosphatebuffered saline containing 1 mM magnesium chloride and 1 mM calcium chloride (PBMC). Sections were rinsed briefly and then incubated for 60 min at room temperature with mouse monoclonal antibody 22/G9, diluted l/100-200 in PBMC. After rinsing in PBMC for 10 min, sections were incubated in affinity-purified, fluorescein-conjugated goat anti-mouse IgG antibody in PBMC for 30-60 min at room temperature in the dark. Slides were coverslipped with Immu-Mount (Shandon) and examined by epifluorescence microscopy. Controls included (1) incubation of sections with secondary antibody alone and (2) incubation of sections in normal mouse serum followed by secondary antibody. 3. Results The retinopathy induced by sodium iodate in pigmented rabbits is manifest initially as necrosis of the RPE, which begins as early as 6 hr after administration of sodium iodate (unpubl. obs. ; see Anstadt
157
et al., 1982). Necrosis of the pigment epithelium is fulminant within 1 day of administration of iodate and is followed several days later by atrophy of the photoreceptors (e.g. shortening of the outer segments). We report observations made from 1 week to 1 month after iodate administration, since subretinal scars in varying degrees of formation are scattered through the fundus. Light Microscopy By l-2 weeks after administration of sodium iodate, scars form where the subretinal space is vacant due to photoreceptor atrophy and removal of the RPE by macrophages [Figs l(A)-(C)]. At these sites the outer nuclear layer and ELM often appear scalloped due to formation of photoreceptor rosettes [Figs l(A) and (B)]. One month after iodate adminstration, rosettes are no longer present and numerous well-developed subretinal scars occupy the tissue space formerly occupied by the RPE and photoreceptors [Fig. l(D)]. Immunostaining for the antigen detected by monoclonal antibody 22/G9 (see Materials and Methods) was localized to the apices of Miiller cells in normal rabbits and, in rabbits that received sodium iodate, wherever the ELM was intact, i.e. in remnant normal retina and in rosettes [Figs 2(A) &d (B)]. The staining disappeared where the ELM had broken down [arrows in Fig. 2(B)]. In sections stained with toluidine blue these sites corresponded to regions where the ELM was disrupted due to photoreceptor atrophy and subretinal scar formation, e.g. the arrow in Fig. l(A) and the zone between the rosettes seen in Fig. l(B). In sections examined by transmission electron microscopy these sites corresponded to areas where the Miiller cell ascending processes had penetrated beyond the ELM and into the subretinal space [Fig. 3(A)].
FIG. 2. Immunofluorescence for Miiller cell microvillar antigen detected by monoclonal antibody 22/G9, from a rabbit that received sodium iodate 3 weeks before being killed. A, In area of normal retina the ELM stains intensely. It appears ragged due to staining of individual microvilli. x 250. B. Where the ELM is disrupted (arrows) due to subretinal scar formation, immunoreactivity
is absent. The ELM encircles the lumina
of rosettes. x 250.
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FIG. 3. Electron micrographs from thin sections through the edge of a scar, examined 2 weeks after iodate. The locale correspor Ids to the edge of the area denoted by the arrow in Fig. l(A). A, A Miiller cell ascending process (P) sends .several branches I (straight arrows) into the scar while another branch (curved arrow) still contributes to the external lir niting membrar le [detailed in (B)]. The scar processes are irregular in profile and form endfeet at the former RPE basement mem lbrane (arrowhe :ads, detailed in inset). Several photoreceptor somata (n, a nucleus) are seen within the scar. IS denotes inner seg ments of atroph :ic photoreceptors. Cell at left is probably a macrophage, as melanosomes apparently within phagosomes were s’een in the part (If the cell outside this field. x 7900. Inset, Detail of endfoot of RPE basement membrane. Plasma membrane here forms
DULLER
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STRUCTURE
FIG. 4. Remant zonulae adherentes and microvilli along the length of scar processes, seen in a thin section (A) and a computer reconst~ction of serial thick sections ex~ed by HW (B). A, Two adjacent scar processes (a light one and a dark one-otherwise similar in their cytology) are connected by xonulae adherentes (za) of the external hmtting membrane type (note extensive cytoplasmic density and focal appositions of plasma membrane) with a few microvilii (arrow). The processes contain abundant glycogen granules and intermediate iYaments. Scars often contain Miiller cell processesof differing electron densities, perhaps related to their different content of intermediate filaments. x 40 600. B, Computer reconstruction of ascending Miiller cell process in a series of O-S-pm thick sections through a scar examined by HYEM, from a rabbit obtained 6 weeks after iodate. The process abuts the basement membrane formerly occupied by the RPE, at top of picture. Along the length of the process two clusters of microvilli [denoted as a solid light zone, since every ~cro~us cannot be traced ; the space between the tracing of the microvillar xone and the tracing of the scar process is due to inaccuracy (e.g. hand movements) during tracing of the structures] are adjacent to external limiting membrane xonulae adherentes, denoted by white bars beneath plasma membrane.
The changes that ascending Miiller cell processes undergo during scar formation were studied in thin sections where the normal ELM was contiguous with a site of scar formation. Here, normal ascending processes con~but~g to subret~al scars arose in proximity to each other, sometimes from the same cell, and could be compared [Fig. 3(A) and (B)]. The Muller cell ascending processes still contributing to ELM were slender, contained numerous ~~chondria, had apical microvilli and formed zonulae adherer&es that consti~te the ELM [Fig. 3(B)]. Those processes contributing to the scar, however, had transformed
contained
into stout, irregular
abundant
intermediate
processes
that
filaments and gly-
cogen granules, and formed endfeet with attachment sites on the remnant RPB basement membrane [Fig. 3(A) and inset]. Apparent remnants of ELM junctional
complexes and associated microvilli were observed along their length in thin sections and in reconstructions of serial thick sections examined by HVEM [Fig. 4(A) and (B)]. The shape of the scar processes varied in younger and older scars [e.g. those seen in Figs l(C) and (D). respectively]. ~mputer reconst~ctions of three ascending processes in a forming subretinal scar revealed a more irregular surface contour than in three reconstructions of ascending processes from an older scar [cf. Figs S(A) and (B)]. This correlated with the fine structure of over 50 scars examined [e.g. Fig. 3(A) from a young scar, and Fig. S(C) from an old scar]. Contact with the RPE basement membrane seemed to influence the shape of the Miiller cell ascending process. In the process illustrated in the stereo pairs in Fig. 6(A), several appendages are seen on the part of the process nearest the viewer, which is not in contact
attachment sites (arrowheads) and has an underlying meshwork of fine filaments. The cytoplasm contains abundant glycogen granules and interme~ate filaments, detailed in Fig. 4(A). From tissue mordanted with tannic acid. x 50000. B, Detail of portion of Miiller cell process seen at curved arrow in (A), from an adjacent section. The process contains numerous mitochondria and still possesses microvilli (arrows) and zonulae adherentes (za) that constitute the external limiting membrane. Scar processes [e.g. those seen in (A)] generally contain fewer mitochondria but more intermediate filaments and glycogen. x 19600.
160
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ET AL
FIG. 5. Computer reconstructions of scar processes in a set of 1-ym thick sections examined by HVEM, from a rabbii t obtained week .s after iodate. (A) is from a young, or early. scar [e.g. processes similar to those seen in Figs l(C) and 3(A)]. ( B) is from a more advanced scar [e.g. processes like those seen in Fig. l(D)]. The stereo electron micrographs in (C) are printed I from the stereo Inegatives of the first section of the series reconstructed in (B). A. This reconstruction is tilted so that the irregu liar shape of the Ejrocess is seen to advantage, e.g. the irregular, smaller process that arises off the right side of the larger one (at the black arrow) and curves downward to contribute to the external limiting membrane, its junctions indicated by the white bar s beneath 6
MijLLER
CELL
STRUCTURE
161
with the basement membrane. However. when the reconstruction is ‘ split ’ [Fig. 6(B)] to reveal the part of the process actually in contact with the basement membrane [and buried in the stack of sections of the reconstruction in Fig. 6(A)], the loss of appendages at this site becomes apparent. The somata and descending processes of Miiller cells at sites of subretinal scar formation appeared normal except for apparent hypertrophy of the foot processes at the inner limiting membrane [e.g. Fig. l(D)]. A detailed analysis of these parts of the Miiller cell, however, was not undertaken, as the most striking changes occurred in the ascending processes. 4. Discussion During
subretinal
scar formation,
the ascending
Miiller cell processes move into the subretinal space and occupy the tissue volume formerly occupied by photoreceptors, interphotoreceptor matrix and pigment epithelium.
Eventually
the processes
reach the
pigment epithelium basement membrane, where they form a glia limitans consisting of glial endfeet lie those abutting retinal blood vessels and the inner limiting membrane (cf. Fig. 11-9 of Hogan, Alvarado and Weddell, 1971). In the interim between moving from the level of the ELM to the basement membrane of the pigment epithelium, the Miiller cell plasma membrane specializations change. While new attachments to the RPE basement membrane form, the microvilli and inFIG. 6. HVEM computer reconstruction of a scar process that runs tangential to the RPE basement membrane. The orientation is 90” clockwise from the reconstructions seen in Fig. 5. A, In the stereo reconstruction the process bears many finger-like processes directed towards the RPE basement membrane (linear profiles at left). B, The same reconstruction seen in (A) is now presented as a stack profile instead of in stereo. The stack is ‘split ’ so that the series of sections through the portion of the process actually contacting the RPE basement membrane (on left) and not observed in the stereo reconstruction in (A), can be compared with the series of sections not bordering the membrane (on right) but which are seen in the reconstruction in (A). Note that the surface of the process contacting the basement membrane is smooth, while that not bordering it has numerous finger-like projections. These latter hid the flat area bordering the basement membrane in the stereo reconstruction seen in (A).
tercellular junctional complexes located at the ELM become scattered over the surface of the ascending process and are eventually lost, Coincident with ELM breakdown and loss of microvilli is the loss of a specific microvilli-associated glycoconjugate recognized by an antibody designated 22/G9 (Pratt, Johnson and Hageman, 1987; Pratt, Hageman and Johnson, 1988). The loss of reactivity for this antigen accom-
panies the loss of cytological polarity of the Miiller cell ascending process during scar formation. This loss of staining may be due to several factors, e.g. diffusion of the antigen through the plane of the plasma membrane to such an extent that it is no longer detected by the procedure used, internalization and degradation of plasma membrane, or loss of epitopes during gliosis. Clearly this phenomenon warrants further study as a
the plasma membrane. The space intervening between these junctions and those of a portion of an adjacent scar process (white arrow) was occupied by the inner segment of an atrophic photoreceptor. This was not included in the reconstruction because it would have obscured reconstruction of the glial processes. Note that the surface facing the RPE basement membrane (linear profiles at top) is flat. B, Reconstruction of a process from an older scar in the same animal as that depicted in (A) [e.g. those seen in Fig. l(D)]. Compared to the process reconstructed in (A), this one is more regular and does not contribute to the external limiting membrane. An electron micrograph of one of the thick sections used to reconstruct this process is seen in (C). C, Stereo HVEM picture of the first l-pm thick section used to make the reconstruction in (B). Note how the reconstruction from the subsequent series of sections has provided more information than is present in this single section, e.g. the extent of the two small processes that surround an adjacent photoreceptor soma (s) and how discrete endfeet are formed at the RPE basement membrane at the top of the picture. x 3500. 11
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mechanism of remodeling the Miiller cell plasma membrane during scar formation. One advantage of the sodium iodate retinopathy model in rabbits is that the pigment epithelium does not remain intact, providing an opportunity to examine the response of the Miiller cell processes to contact with a novel basement membrane. To this extent the model tests the hypothesis that at sites where Miiller cells form a glia limitans (e.g. at the inner limiting membrane of the retina or around retinal blood vessels) a basement membrane is always present and provides cues for the formation of glial endfeet and their cytologic specializations, such as attachment sites. When brought into contact experimentally with the RPE basement membrane, Miiller cell endfeet identical to those at retinal blood vessels and the inner limiting membrane are observed. The plasma membrane in contact with basement membrane seems to respond in two ways: it forms attachment sites, and it loses the uneven contour seen in Miiller cell plasma membrane that does not appose the basement membrane. This contact seems to precede, and perhaps signal shape changes in the rest of the process, for those processes containing the basement membrane seem more uniform in contour than those that do not. Although the signal molecules involved in eliciting these changes have not been identified, it is possible that basement membrane constituents such as fibronectin or laminin are involved. The responsiveness of glia to these molecules and several growth factors that may be present during subretinal scar formation (e.g. fibroblast growth factors from macrophages) has been documented (see Harvey, Roberge and Hjelmeland, 1987). The rabbit model used in this study may be suitable for identifying those factors responsible for controlling the differentiation of the Milller cell and its ‘redifferentiation ’ during subretinal scar formation. Acknowledgements Supported by NE1 grants EY08284 (G.E.K.) and EY06463 (G.S.H.) ; unrestricted departmental grants from Research to Prevent Blindness, Inc to the Department of Ophthalmology, Albert Einstein College of Medicine and to the Bethesda Eye Institute ; and a grant from NIH (RR01 12 19) supporting the Biological Microscopy and Image Reconstruction Resource located at the New York State Department of Health in Albany, New York. References Anstadt, B., Blair, N., Rusin, M., Cunha-Vaz, J. and Tso, M. (1982). Alteration of the blood-retinal barrier by sodium iodate: kinetic vitreous fluorophotometry and
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ET AL.
horseradish peroxidase tracer studies. Exp. Eye Res. 35. 653.
Bernstein,P., Carlson.A. and Bok, D. (1991). The retinyl ester synthetase is a cellular target for the retinal pigment epithelium toxin sodium iodate. Invest. Ophthalmol.Vis. Sci. (Suppl.)32, 1250. Erickson,P., Fisher,S..Anderson,D., Stern,W. andBorgula, G. ( 1983). Retinaldetachmentin the cat: outer nuclear and outer plexiform layers.Invest. Ophthalmol. 24. 92 7. Glusman.S.,Korte, G. (1988). Miiller cellresponseand scar formation during sodiumiodateretinopathy in rabbits. Invest. Ophthalmol. Vis. Sci. (Suppl.)29, 289. Graymore, C. (1970). Biochemistry of the retina. In Biochemistryof the Eye. (Ed. Graymore. C. N.) Pp. 645-735. AcademicPress:New York. Harvey, A., Roberge. F. and Hjelmeland, L. (1987). Chemotaxisof the rat retinal glia to growth factors found in repairing wounds.Invest. Ophthalmol. Vis. Sci. 28, 1092-g. Hogan,M., Alvarado, J. and Weddell,J. (1971). Histology of the HumanEye. W. B. Saunders:Philadelphia. Ikui, H. (1974). Morphology and pathology of the retinal glia. Acta Ophthalmol. (Jap.) 75, 1245. Ishikawa, Y. (1974). Histological studies of repairing processes after xenon photocoagulationin the monkey retina. I. Electron microscopic studies on cellular responses in the early repairing stage.Acta Ophthalmol. (Jap.)78, 606. Johnson, L. V. and Blanks, J. C. (1984). Application of acrylamideasan embeddingmediumin studiesof lectin and antibody-bindingin the vertebrate retina. Curr. Eye Res. 3, 969-74.
Korte, G. E. (1990). High voltage electron microscopy of reactive Mtiller cellsin the rabbit retina. Anut. Rec. 226, 52A. Korte, G. E., Reppucci, V. and Henkind, P. (1984). RPE destruction causes choriocapillaris atrophy. Invest. Ophthalmof. Vis. Sci. 25, 1135. Laqua. H. and Machemer,R. ( 1975). Glial cell proliferation in retinal detachment (massive periretinal proliferation). Am. J. Ophthalmol. 80, 602. Lewis,G.. Erickson,P., Guerin. C., Anderson,D. and Fisher, S. (1989). Changesin the expressionof specificMiiller cell proteinsduring long term retinal detachment.Exp. Eye Res. 49, 93.
Marko, M.. Leith, A. and Parsons, D. (1988). Threedimensionalreconstructionof cellsfrom serialsections and whole-cellsmounts usingmultilevel contouring of stereo micrographs. 1. Electron Microscopy Tech. 9, 393.
Pratt, D. V., Hageman, G. S. and Johnson,L. V. (1988). Immunologicalcharacterizationof an antigenexpressed by Miiller cell apical microvilli. Invest. Ophthalmol. Vis. Sci. (Suppl.) 29, 244. Pratt. D., Johnson, L. V. and Hageman, G. S. (198 7). Polarizeddistribution of a Miiller cell-specificantigen. Invest. Ophthalmol. Vis. Sci. (Suppl.)28, 258. Pratt, D.. Korte, G. E., Johnson,L. V. and Hageman.G. S. (1988). Altered expressionof a Miiller cell-specific antigen in sodium iodate induced retinopathy. Eight International Congresson EyeResearch.Proc. Intl. Sot. Eye Res. 5, 124.
