THE JOURNAL OF COMPARATIVE NEUROLOGY 312~175-192 (1991)

Intraorbital Transection of the Rabbit Optic Nerve: Consequences for Ganglion Cells and Neuroglia in the Retina JURGEN SCHERER AND JUTTA SCHNITZER Max-Planck-Institut fur Hirnforschung, Abteilung Neuroanatomie, W-6000 Frankfurt am Main 71, Federal Republic of Germany

ABSTRACT Rabbit retinae were stained with antibodies to glial fibrillary acidic protein (GFAP) at various times up to 5 months after complete unilateral intraorbital optic nerve transection, which is known to induce degeneration of ganglion cell axons and perikarya in the retina. A transient immunoreactivity for GFAP was observed in Muller glial cells that normally lack this marker. Muller-cell GFAP immunoreactivity became detectable 4 days after the lesion, but Miiller cells were no longer labeled 3 months later. GFAP-labeledastrocytes located in the nerve fiber layer showed no change in immunoreactivity at any stage after transection. Application of horseradish peroxidase to the left and right superior colliculus of a rabbit whose optic nerve had been transected unilaterally 2 years before confirmed the completeness of the transection. Yet electron microscopy showed the presence of some healthy-looking ganglion cell axons in the lesioned retina, although these cells were deprived of their target. Labeling retinal wholemounts with neurofilament antibodies confirmed the presence of some ganglion cell axons and perikarya in the retina more than 2 years after transection. The course of these axons suggested that they were remnants of axons. Using antibodies t o galactocerebroside (GC) we found that, as in the normal rabbit, these persisting ganglion cell axons were myelinated in the medullary rays. Although many ganglion cell axons had disappeared after 2 years, the number of neuroglial cells (includingastrocytes and oligodendrocytes) present in the medullary ray region was not altered. The cell bodies of some oligodendrocyteswere covered with a myelin sheath, an aberrant feature not seen normally. Key words: axotomy, neuronal degeneration,astrocytes, Miiller cells, oligodendrocytes

Lesioning of the optic nerve of adult mammals has long been known to lead to degeneration of the axons in the nerve. In addition, ganglion cells residing in the retina begin to degenerate. Crushing the optic nerve of mice induces the loss of about 80%of the large and medium-sized (ganglion) cells within 10 days (Allcutt et al., '84a). The question of whether all ganglion cells disappear-after long survival times-remains controversial, After the same kind of lesion, namely intracranial optic nerve transection in cats, the survival of some scattered ganglion cells was reported about 15.5 months later (Hollander et al., '851, while another group claimed the total loss of ganglion cells fourteen months after transection (Wassleet al., '87). We have shown recently that microglial cells residing in the retina respond to optic nerve transection by increasing in number in the inner plexiform and nerve fiber layers (Schnitzer and Scherer, '90). In both strata degenerating ganglion cell axons and dendrites are present and require removal. In addition, the enzyme nucleoside diphosphatase, which is a normal constituent in Golgi apparatus and O

1991 WILEY-LISS, INC.

lysosomal membranes (Novikoff and Goldfischer, '61) but also in microglial cell plasma membranes (Murabe and Sano, '821, increases its activity in response to injury, suggesting increased phagocytotic activity. This increase in cell number and enzyme activity is transient. By 5 months postlesion, both their number and their enzyme activity have returned to approximately normal levels. On these grounds, we have suggested that by this time, the degeneration of ganglion cells has ceased (Schnitzer and Scherer, '90). The nerve fiber layer at the medullary ray region of the rabbit is a region where, in the normal adult rabbit, ganglion cell axons become myelinated by oligodendrocytes before entering the optic nerve, and a region to which nerve fiber layer astrocytes are confined (see Schnitzer, '85). In the present study we asked: how do neuroglial cells in this region respond to the degeneration and disappearance of ganglion cell axons? Will the number of astrocytes remain Accepted June 12,1991

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unchanged when the ganglion cell axons disappear following nerve crush [as described by Allcutt et al. (’84a)l,or will their number decline [as described for a retina whose axons had degenerated following photocoagulator lesions applied to the retina (Karschin et al., ’86b)]? Will the number of oligodendrocytes remain the same or decrease in the medullary ray region when the ganglion cell axons disappear? We approached these questions by comparing the staining pattern of antibodies to glial fibrillary acidic protein (GFAP) and galactocerebroside (GC), antibodies which mark astrocytes and oligodendrocytes, respectively, in “lesioned” and control retinae. We also evaluated semithin sections prepared from various parts of the medullary ray region. We observed that astrocytes failed to acquire stronger GFAP labeling in the lesioned retina than in the normal retina, but Muller glial cells transiently expressed GFAP immunoreactivity for a few weeks after lesion. More than 2 years after transection the number of astrocytes and oligodendrocytes in the medullary rays was almost identical to the number in normal retina. Using antibodies to neurofilament protein, which label ganglion cell axons in the retina (Drager et al., ’841, we will show that even 2 years after cutting the optic nerve some ganglion cell axons were stained in whole-mounted retinae. We therefore wished to confirm that there was some ganglion cell survival, although the respective optic nerve fibers were indeed completely transected intraorbitally. To do that we applied horseradish peroxidase (HRP) to both superior colliculi to which almost all ganglion cells present in the rabbit retina are known to project (Vaney et al., ’81b). We will describe that HRP accumulated in ganglion cells of the nonlesioned but not in the lesioned retina. The same ‘‘lesioned’)retina contained, however, viable ganglion cell axons, as demonstrated at the electron microscopic level, which were myelinated in the medullary ray region. We will suggest that some ganglion cells are able to survive without their target for more than 2 years, a phenomenon which is not yet understood.

MATERIALS AND METHODS Transection of the optic nerve Adult New Zealand white rabbits were used for the present study. Under deep anesthesia (30 mg/kg Ketanest; 10 mg/kg Rompun) the left optic nerve was approached intraorbitally. Care was taken not to disturb the integrity of the intraretinal blood supply. This was achieved by cutting the nerve more than 1 mm behind the bulbus, since the central artery of the retina enters the optic nerve about 1 mm behind the entrance of the nerve into the sclera (Davis, ’29). The transection was performed unilaterally, the right retina serving as a control. Rabbits (number of animals in parenthesis) treated in the above manner were allowed to survive for postoperative periods of 2 (31, 3 (11, 4 (31, 7 (41, 12 (l),14 (l), 21 (l),28 (l), and 84 (1)days, and 5 (l), 9 (l), 16 (11, and 25 (1)months, respectively.

Tissue preparation After the prescribed survival time, animals were killed by an overdose of Nembutal. After enucleation, the cornea, lens, and vitreous body were removed and the remaining eyecups were immersion-fixed for 2 hours at room temperature in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2 (PB). Some eyecups were divided vertically from supe-

rior to inferior through the optic disc. The resulting retinal halves were then treated differently, one half being fixed for immunocytochemistry, and the other (for ultrastructural studies) fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. In these cases, for comparative reasons, the nasal and temporal halves of the retinae from the same animal were always treated identically. Retinae to be immunolabeled as retinal wholemounts were removed from the eyecup and washed overnight in PB. Retinae used for cutting vertical sections were soaked with 30% sucrose. Vertical cryostat sections (16 pm thick) were prepared as described (Schnitzer, ’85). Retinal eyecups fixed in glutaraldehyde were washed in cacodylate buffer. The retinae were peeled out and a horizontal strip (about 2 mm wide, 10 mm long) was cut out beginning at the optic disc and including most of the medullary ray region. These strips were divided into ten consecutive squares (2 x 1mm) and numbered. The tissues were osmicated, dehydrated, and embedded in Epon. Vertical, semithin 1km thick sections were counterstained with toluidine blue. Ultrathin sections were counterstained with lead citrate, and examined in an electron microscope (Zeiss EM 10).

Evaluation of neuroglial nuclei in semithin sections Semithin vertical sections through the medullary rays were evaluated for the presence and number of nerve fiber layer neuroglial cells. Sections from lesioned and control retinae obtained at equal distances from the optic disc were compared. Entire semithin sections, which were up to 2 mm long, were drawn by means of a drawing tube at a final magnification of X400. At this magnification the number and position of all neuroglial cell nuclei residing in the nerve fiber layer can be determined. Darkly and lightly Nissl-stained nuclei were included in the counts. No attempt was made to distinguish between astrocytes and oligodendrocytes. Darkly granulated irregular shaped nuclei, which most likely represent microglial cells, were excluded from the counts.

Antisera Monoclonal mouse antibodies directed against GFAP (clone G-A-5) and mouse antibodies directed against vimentin (clone V9) were obtained from Boehringer (Mannheim, F.R.G.) and used at a dilution of 5 kg/ml. Monoclonal mouse antibody to the human 70 kD neurofilament protein (NF70; clone NFZF11) was obtained from Biochrome (Berlin, F.R.G.) and used at a dilution of 1 : l O . Monoclonal mouse antibodies to galactocerebroside were a gift of Dr. B. Ranscht and were diluted 1:20. Fluorescein isothiocyanate (F1TC)-coupled goat anti-mouse immunoglobulins were purchased from Cappel Laboratories and used at dilutions of 1 5 0 . Rabbit anti-mouse immunoglobulins were obtained from Miles Scientific and diluted 1:50; monoclonal mouse peroxidase antiperoxidase complex (mouse PAP; Sternberger-Meyer, Immunocytochemicals, Inc.) was used at a dilution of 1:lOO.

Immunocytochemical procedures Retinal wholemounts. The antigens were visualized using the peroxidase-antiperoxidase method according to Sternberger (’79). The procedure has been described in detail (Schnitzer and Karschin, ’86). Briefly, free-floating

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Fig. 1. Whole-mounted rabbit retina labeled with antibodies against GFAP at various days (a,4 days; b, 7 days; c, 21 days; d, 84 days) after optic nerve transection. Muller cells of the rabbit retina are transiently GFAP immunoreactive. The micrographs were taken in the inferior

retina which is astroglia-free. The focus is on the inner plexiform layer, and radially oriented Muller cell processes are seen as round or oval structures using Nomarski optics. Scale bar = 50 bm.

retinae were incubated with the first antibody (monoclonal antibody directed against GFAP, vimentin, and NF70, respectively) and incubated with rabbit anti-mouse immunoglobulins, followed by mouse PAP. Each incubation step was carried out for 2-4 days at 4°C. Antibody binding was visualized with 3,3'-diamino benzidine tetrahydrochloride. Retinae were flat-mounted, ganglion side up, according to Wassle et al. ('75).

Histological sections. The presence of astrocytes, oligodendrocytes, and ganglion cell axons was verified in frozen vertical sections which were taken close to the optic disc. These sections contained parts of the superior retina, the myelinated region, and the inferior retina. Sections were stained by the indirect immunofluorescence technique. The procedure was the same as described (Schnitzer, '88b). In brief, sections were incubated with the first antibody

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Fig. 2. Whole-mounted rabbit retina, inferior, outside the medullary rays. Focus is on the inner plexiform layer. a: Two weeks following optic nerve-cut vimentin antibodies labeled Muller cell processes, but

no further cellular elements. b: Microglial cells labeled enzyme histochemically for nucleoside diphosphatase (NDPase). Cells stratifying in this layer have radially oriented ramified processes. Scale bar = 50 pm.

(directed against GFAP, galactocerebroside, and NF70, respectively), followed by the fluorescent second antibody. Sections were mounted with buffered glycerol.

mounted ganglion cell side up as described. The intracranial parts of the left and right optic nerves of the “lesioned” rabbit were fixed for 2.5 hours in 2% glutaraldehyde in PB, washed, cut into 1mm long pieces, osmicated, dehydrated, and embedded in Epon.

Retrograde labeling of ganglion cells Two adult New Zealand white rabbits were used. One rabbit had survived the transection of its left optic nerve for 25 months, the second rabbit had not been operated and served as control. The animals were deeply anesthesized with intraveneous administration of sodium pentobarbital (20 mg/ml) and then placed in a stereotaxic headholder. The position of the rabbit superior colliculus was estimated from an atlas of the rabbit diencephalon (Sawyer et al., ’54). In accordancewith these coordinates, the skull was trepaned on each side, dorsal to the superior colliculus. By means of a Hamilton syringe, five 1 111 injections of HRP (Sigma Munich, P-8375; 30% in 2% dimethylsulfoxide) were applied to each superior colliculus [coordinates, right superior colliculus posterior (p.) 9 mm, lateral (1.) 2.6, 3.6, and 5.2 mm; p. 11.5 mm, 1. 2.8 and 5.0 mm; left superior colliculus p. 9 mm, 1. 2.4 and 5.7 mm; p. 10.5 mm, 1. 4.0 mm; p. 11.5 mm, 1. 2.8 and 4.7 mm; depths 4 mm at each injection site]. Care was taken to place the injections as symmetrically as possible. The presence of heavy marking in the control retina confirmed correct placement of the HRP injections in the optic nerve-sectionedanimal. The animals were allowed to survive for 24 hours and were then killed by an overdose of Nembutal. After enucleation, the retinal eyecups were fixed for 30 minutes with 2% glutaraldehyde dissolved in 0.1 M PB, pH 7.2. After washing with PB for 2 hours, the retinae were isolated. The HRP retrogradely transported to retinal ganglion cells was visualized by incubating the free-floating retina in 0.05% 3,3’diaminobenzidinediluted in PB for 15 minutes, followed by an incubation, including hydrogen peroxide, at a final concentration of 0.01% for 30 minutes. Retinae were

RESULTS Neuroglial cell markers in the normal and lesioned retina Muller glial cells transiently express GFAP. The degeneration of ganglion cells starts a few days after intraorbital transection of the optic nerve (Schnitzer and Scherer, ’90) and by 3 months many ganglion cell bodies and axons have disappeared. In the normal rabbit retina antibodies to GFAP stained astroglial cells only, which are located in the nerve fiber layer of the medullary ray region. With the antibody and the procedure used, Miiller glial cells were GFAP negative (Schnitzer, ’85; Schnitzer and Karschin, 1986). Following transection of the optic nerve, Miiller glial cells transiently acquired GFAP immunoreactivity. This finding is illustrated in Figure 1, using micrographs taken from the astroglia-free “periphery” of a retinal wholemount to dem-

Fig. 3. GFAP-labeled whole-mounted rabbit retinae. a: Lesioned retina, 3 months after optic nerve cut. b: Control retina. The micrographs were taken at the edge between the astroglia-bearing, GFAPpositive medullary rays (bottom) and the astroglia-lacking, peripheral inferior retina (top). In the control (b) and in the lesioned (a) retina many GFAP-labeled astrocytic processes are aligned in parallel with ganglion cell axons which are visible as smooth stripes using Nomarski optics. In the control retina (b) the ganglion cell axon bundles are numerous, and in the lesioned retina (a) they are considerably fewer. Scale bar = 100 pm.

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onstrate the effect better. Note that by focusing on the inner plexiform layer, radially oriented Muller cells are visible as round structures which can easily be detected even without any labeling when Nomarski optics are used. Few Muller cells were GFAP positive 4 days after transection (Fig. la). Their number increased gradually thereafter (Fig. lb; 7 days after transection). About 3 weeks after transection, some Muller cells had lost GFAF’ immunoreactivity (Fig. lc), and by 3 months none were visibly stained (Fig. Id). When we treated control and lesioned retinae with antibodies t o another intermediate filament protein, vimentin, which is a well-established marker for mammalian Muller cells (see Schnitzer, ’88a, for review), we could not observe any alteration with respect to the staining intensity of Muller cells in any region of these cells and at any survival times studied. Microglial cells have been reported to express the intermediate filament protein vimentin in response to injury (Graeber et al., ’88). We therefore carefully inspected lesioned retinae for traces of vimentin labeling which might be located in microglial cells. Figure 2a shows vimentinstained profiles in a retinal wholemount 2 weeks after nerve cut. The focus is on the inner plexiform layer. All labeled profiles resemble the radially oriented Muller cell processes (compare to Fig. 1).Microglial cells located in the inner plexiform layer have a star-shaped morphology with many fine, delicate processes (Fig. 2b; nucleoside diphosphatase, NDPase, labeling; for details see Schnitzer, ’89).Cells with this kind of morphology were never seen to be vimentin positive at any survival time. The same holds true for NDPase-labeled microglial cells situated in the nerve fiber layer. In this layer, only Muller cell endfeet and astrocytes are vimentin positive (not shown). Thus we concluded that microglial cells do not acquire detectable amounts of vimentin protein in response to injury. GFAP-positive astrocytes. When we compared the Fig. 4. GFAP-labeled vertical sections through the medullary ray GFAP labeling of astrocytes seen in whole-mounted control retinae with that seen in “degenerating” retinae, no major region. a: Lesioned retina 5 months after optic nerve cut. b: Control GFAP-labeled astroglial cell processes are densely packed in the differencewas observed at survival times between 2 days to retina. rather thin nerve fiber layer (NFL) of the lesioned retina (a); they are 3 months. Only a few stellate-shaped GFAP-positiveastro- less dense in the control retina (b). ONL, outer nuclear layer; OPL, cytes, which were in contact with blood vessels, possessed a outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform stronger GFAP-labeling intensity than the cells in the layer; GCL, ganglion cell layer. Scale bar = 50 pm. normal retina. “Reactive” gliosis, i.e., enhanced GFAF’ staining of astrocytes, was not observed. By 3 months after transection, at a time when many processes were densely packed in the much thinner nerve axons have disappeared, astroglial cell processes still follow fiber layer of the lesioned retina (Fig. 4a; 5 months after the course of the remaining axon bundles. This is most transection). Galactocerebroside-labeled oligodendrocytes. The rabevident at the border between the astroglia-bearing medullary ray and the astroglia-free “periphery” (Fig. 3a). For bit is the only known mammalian species in which the comparison,the same area of a GFAP-labeled control retina axons of ganglion cells become myelinated by oligodendrois shown where the ganglion cell axon bundles are clearly cytes already in a small region within the retina, the visible as smooth stripes (Fig. 3b). At all survival periods medullary ray region (Berliner, ’31; Davis, ’29; Narang and studied, GFAF’-positiveastroglial cells remained restricted Wisniewski, ’77; Schnitzer, ’85). By applying antibodies to the medullary ray region, where they occupied the same directed against the oligodendrocyte-specific marker GC to proportion of the retina as in the nonlesioned animal. Thus, vertical sections through the medullary rays, we observed the degeneration of ganglion cells is accompanied by neither that the thinner nerve fiber layer of the lesioned retina was an obvious morphological nor immunocytochemical alter- GC positive by 5 months after nerve cut (Fig. 5a), although its staining intensity was somewhat weaker than in the ation of astrocytes. The reduced thickness of the nerve fiber layer which control retina (Fig. 5b). This observation suggested that accompanies the degeneration of ganglion cells leads to a either some myelinated ganglion cell axons, or at least modified appearance of GFAP-labeled astroglial cell pro- myelin debris, are still present by 5 months after transeccesses in vertical sections. Compared to the normal retina tion. We will demonstrate below that a substantial propor(Fig. 4b), where the network of labeled astroglial processes tion of ganglion cells survive the transection, and their is rather loose, numerous GFAP-positive astroglial cell axons are myelinated in the medullary ray region.

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sections through ~ i 5, ~ Galactocerebroside , (GC)-labeled the medullary ray repion, a: Lesioned retina 5 months after optic nerve cut. b: Control retina. The nellre fiber layer (NFL) ofthe lesioned retina (a) is thinner and less intensely GC-labeled to the control retina (b), F~~abbreviations, see ~i~~~ 4 legend, scalebar = so I*m.

Evidence that ganglion cell axons persist in retinae after complete optic nerve transection Neurofilament labeling in the retina after intraorbital optic nerue cut. By staining vertical sections with antibodies to NF70, which is known to label mammalian ganglion cell axons, but also some amacrine cells, and the axonless A-type horizontal cell (Drager et al., ’841,we were able to confirm that some ganglion cell axons were still present in the nerve fiber layer (Fig. 6) 5 months after transection. Note that amacrine cells (stratifying in the inner plexiform layer) and horizontal cells (stratifying in the outer plexiform layer) were also detectably labeled by antibodies against NF70 in both the lesioned and control retinae. NF70 staining of vertical sections prepared at various survival stages following optic nerve transections revealed that some ganglion cell axons persisted for more than 2 years, which was the longest survival time studied so far. The majority of the NF7O-positive axon bundles which are detectable in lesioned rabbit retinal wholemounts follow the same direct course towards the optic disc as they do normally. In both the lesioned and the control retina, the axon bundles run rather parallel from the retinal periphery

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Fig. 6. Neurofilament (NF7OI-labeledvertical sections through the medullary ray region. a: Lesioned retina 5 months after optic nerve cut. b: Control retina. In the lesioned (a) and in the normal retina (b) NF70-positive fibers are seen in the outer plexiform layer (OPL), the inner Plefiform layer (IPL), and in the nerve fiber layer (NFL). Fewer NF7O-immunoreactive fibers are detectable in the NFL of the lesioned retina (a) than in the control retina (b). For other abbreviations, see Figure 4 legend. Scale bar = 50 Fm.

towards the center where they bend to approach the optic disc almost horizontally (see the schematic illustration in Fig. 7 ) . At the same retinal eccentricity (dark squares in Fig. 71, the NF-positive axon bundles are always less numerous in the lesioned retina (Fig. 8) than in the control retina (Fig. 91, but the roughly parallel course of fibers is indistinguishable. Very few randomly arranged NF-labeled processes were seen in the lesioned retina in the myelinated region close to the optic nerve head (not shown). The small, round NF7O-positive “profiles” seen in Figures 8 and 9 represent NF70-immunoreactive displaced “coronate” (Vaney et al., ’81a) amacrine cell bodies, whose cellular processes are not in focus. In contrast to these amacrine cell bodies and to ganglion cell axons, ganglion cell somata are barely NF70 immunoreactive unless an appropriate colchicine treatment is performed in situ (Drager et al., ’84). One of the few ganglion cells which were NF70 positive in the lesioned retina (which was not exposed to colchicine) is shown in the montage in Figure 10. One can follow quite easily the course of its axon (Fig. 10, arrows) before it joins an axon bundle. However, the dendritic

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Fig. 7. Schematic drawing of the nasal halves of two retinal wholemounts labeled with antibodies to neurofilament protein (NF70). The lesioned retina was prepared 16 months after optic nerve cut. The lines running from the peripheral retina towards the optic disc show

the course of some NF7O-positive ganglion cell axons. Their course is indistinguishable in the lesioned retina and in the control retina. The dark squares in the inferior retina indicate the position where the micrographs shown in Figures 8 and 9 were taken.

arborization of this ganglion cell is labeled in too rudimentary a manner to classify this cell. The same was true for the few other NF70-positive ganglion cell somata. Thus, classification and quantification of the ganglion cells remaining more than 2 years after optic nerve cut were not possible in NF7O-stained retinal wholemounts. Evidence for complete transection of the optic nerve. The presence of neurofilament-labeled ganglion cell axons more than 2 years after optic nerve cut prompted us to confirm that we had indeed transected the optic nerves completely. This confirmation was performed by two different methods. One approach was to label ganglion cells retrogradely with HRP from one of their targets, the superior colliculus, to which almost all ganglion cells present in the rabbit retina are known to project (Vaney et al., ’81b). Retrograde transport was allowed for 24 hours. In both retinae of a control (nonoperated) rabbit a large number of HRP-labeled cells were found in the ganglion cell layer. In the experimental rabbit, many somata residing in the ganglion cell layer of the control retina revealed HRP labeling but no cell was labeled in the lesioned retina (not shown). This finding confirms the completeness of the transection. Ganglion cell axons degenerate anterogradely to the transection side. We performed a second (ultrastructural) approach to demonstrate that the optic nerve had indeed

been completely transected. We examined the intracranial part of the transected and control optic nerve of the same rabbit that had received injections of HRP into the superior colliculus, which was transported into the control retina but failed to appear in the lesioned retina. The transected optic nerve was much thinner compared to the normal optic nerve (Fig. 11).The latter consists of approximately 394,000 myelinated ganglion cell axons (Vaney and Hughes, ’76). Evaluation of ultrathin sections obtained from the lesioned nerve confirmed that ganglion cell mons have disappeared. Careful examination of entire sections through the lesioned nerve showed that about 20 myelin-like sheaths were found per cross section; some of them surrounded axon-like structures, and others had an empty core (Fig. 12 shows four of these myelin-like structures). The “degenerated” optic nerve consists mainly of astroglial cells, whose cellular processes are loaded with intermediate filaments (Fig. 12, indicated by asterisks), and of oligodendrocytes (Fig. 12, oligo). This finding also confirms the completeness of the transection. Ganglion cell axons persist in the retina after optic nerve cut. We compared semithin vertical sections from control and lesioned retinae prepared from the temporal medullary ray region of the same rabbit whose lesioned retina showed no retrogradely HRP-labeled ganglion cell (see above) and whose ganglion cell axons had completely

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Fig. 8. Neurofilament (NF7Oblabeled ganglion cell axons in a retinal wholemount 16 months after nerve cut. Ganglion cell axons run mostly parallel to each other. This micrograph was taken in the inferior retina (see Fig. 7). The small, round NF7O-positive“profiles” represent immunolabeled displaced amacrine cells. Scale bar = 100 km.

disappeared from the optic nerve. Since in the normal rabbit retina the thickness of the nerve fiber layer in the medullary ray region changes with distance from the optic disc (see Fig. 14,control), we therefore always compared semithin sections from lesioned and control retinae which were taken at the same eccentricity. Many myelinated ganglion cell axons were detected in the thin nerve fiber layer of the lesioned retina (Fig. 13a, lesioned; Fig. 13b, control). At all eccentricities examined, the nerve fiber layer of the lesioned retina is about 50% thinner than normal. (See Fig. 14, camera lucida drawings at three different eccentricities. Each round or oval dot located in the nerve fiber layer represents one neuroglial cell nucleus which will be considered below.) In conclusion, although no ganglion cell was retrogradely labeled with HRP, and although the ganglion cell axons had degenerated anterogradely to the lesion side, myelinated ganglion cell axons were readily detectable in the medullary ray region more than 2 years after the lesion.

Quantification of neuroglial cell numbers in the nerve fiber layer of lesioned retinae Besides myelinated ganglion cell axons, the nerve fiber layer of the medullary ray region contains neuroglial cells. In Figure 13, arrows point to darkly and lightly Nisslstained nuclei which represent astrocytes and oligodendrocytes. No attempt was made to distinguish between these

two types of neuroglial cells. Microglial cells (characterized by darkly granulated, irregular-shaped nuclei) were excluded from the counts. When semithin sections of the medullary rays at various eccentricities from the optic disc were inspected for the presence of nerve fiber layer neuroglial cells we observed that their cell bodies were always more densely packed in the nerve fiber layer of the “lesioned” than in the control retina. This is shown in Figure 14 where each round to oval dot represents one glial cell nucleus. We determined the number of neuroglial cells which were found in the nerve fiber layer within 1mm of lengths in the six sections shown in Figure 14. The results are given in Table 1. The data demonstrate that although many ganglion cell s o n s have disappeared more than 2 years after transection, and although the nerve fiber layer became clearly thinner, the number of neuroglial cells remained roughly constant. This means that the neuroglial cells occupy relatively more space in the nerve fiber layer (their cell bodies become more densely packed). This is in accord with the observation that the GFAP-positive processes form a denser network in the nerve fiber layer of the lesioned retina than in the normal retina (see Fig. 4). Electron microscopy confirmed that the GC immunoreactivity described above is due to surviving myelinated axons in the lesioned retina (Fig. 15). An unmyelinated axon (marked by an asterisk in Fig. 15) is surrounded in a

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Fig. 9. Neurofilament (NF70blabeled ganglion cell axons in a whole-mounted control retina. The micrograph was taken from the inferior retina (see Fig. 7 for position). Ganglion cell s o n s are more numerous than in the lesioned retina (Fig. 8). Scale bar = 100 pm.

tongue-like manner by an oligodendrocytic process. The oligodendrocyte (oligo)shown in this micrograph is covered by a myelin sheath. Many but not all oligodendrocytes present in the lesioned retinae showed this feature, which was never observed in the control retinae. Figure 15 also shows that by 9 months after the cut, myelin breakdown products or degenerating s o n s were rarely seen. Thus the axons which are present after long-term survival form a rather constant population which for unknown reasons is no longer suffering from transection.

DISCUSSION This study has documented that following transection of the optic nerve of adult rabbits many retinal ganglion cells degenerate. This degeneration is accompanied by a transient expression of GFAP by Miiller cells. A proportion of ganglion cells survives the transection, and their axons are myelinated by oligodendrocytes in the medullary ray region.

Immunoreactivity of glial cells following optic nerve transection Muller cells begin to express the intermediate filament protein GFAF' at a time when the first degenerating ganglion cells are detectable. This protein is, at least with the monoclonal antibody and the fixation method we used,

immunocytochemicallynot detectable in Muller cells of the normal rabbit retina (Schnitzer, '85, '88b; Schnitzer and Karschin, '86; Tout et al., '88). In contrast, Vaughan et al. ('90) using different antibodies have shown GFAP-immunoreactive Muller cell endfeet in normal rabbits. The appearance of GFAP in Muller cells in retinae in which neurons are degenerating is in accord with observations following stab wounds, retinal detachment, light damage, or inherited degeneration (Bignami and Dahl, '79; Miller and Oberdorfer, '81; Shaw and Weber, '83; Eisenfeld et al., '84; Erickson et al., '87; Ekstrom et al., '88; Lewis et al., '89). It has been demonstrated that the GFAP expression in Muller cells in response to injury is due to the presence of mRNA encoding for GFAP, a message which is not detectable in "normal" Muller cells (Sarthy and Fu, '89). In our study, GFAP immunoreactivity of Muller cells was transient. By 3 months after transection they are no longer visibly stained. Interestingly, microglial cell responses which occur after optic nerve cut are also over by 5 months after the transection (Schnitzer and Scherer, '90). The stimulus inducing GFAP expression in Miiller cells in vivo is unknown. In the brain, the concentration of GFAP in astrocytes seems to be regulated by glucocorticoids, and a participation of the adrenal gland in astroglial responses to injury has been suggested (O'Callaghan et al., '89). However, in these in vivo experiments the GFAP content was downregulatedafter administration of glucocor-

Fig. 10. Neurofilament (NF'IOblabeled ganglion cell found in a lesioned retina 16 months after optic nerve cut. The axon of this NF-positive ganglion cell runs perpendicular (arrows) to other axon fibers before it joins an axon bundle after several hundred micrometers. Scale bar = 50 km.

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(CONTROL)

immunoreactive (Rieske et al., '89; Scherer and Schnitzer, in preparation).

Ganglion cells persist for more than 2 years The present study has shown that even 2 years following complete intraorbital transection some healthy-looking ganglion cell axons reside in the nerve fiber layer. In contrast, anterograde to the lesion site, close to the chiasma, the ganglion cell axons have completely degenerated. The remarkable resistance of a proportion of ganglion cells to retrograde degeneration (induced by photocoagulation lesions or optic nerve cut) has now been noted in a variety of species (cat: Eysel and Peichl, '85; Hollander et al., '85; 1 Peichl and Eysel, '86; rabbit and guinea pig: Eysel and 500 p m Peichl, '85; mouse: Allcutt et al., '84a,b; Grafstein and Ingoglia, '82; rat: Barron et al., '86; Misantone et al., '84), Fig. 11. Camera lucida drawings of the outlines of semithin sections although their complete disappearance has also been deof a left and a right optic nerve of a rabbit whose left optic nerve had scribed (Wassle et al., '87). been transected intraorbitally 25 months before. The sections of the It has been stated that ganglion cells of all morphological nerves were taken about 2 mm from the chiasm. The lesioned nerve is considerably smaller than the control nerve. types survive in the cat (Eysel and Peichl, '85). Another group reported the preservation of alpha- and gammaganglion cells in the same species (Hollander et al., '85). In ticoids. This finding is in clear contrast to in vitro studies the mouse retina, small ganglion cells seemed to be the least showing that hydrocortisone, as well as other hormones sensitive to axotomy (Allcutt et al., '84a). We will describe and growth factors, induce the synthesis of GFAP in brain in a separate study that about 10% of the ganglion cells astrocytes (Morrison et al., '85). The stimulus which in- survive optic nerve transection a t all retinal eccentricities duces retinal Muller cells to express GFAP will have to be (Schnitzer and Peichl, in preparation). We have shown that, despite the clearly reduced number determined before we understand whether this gene activation might be regulated via cyclic AMP and protein kinase of neurofilament-immunoreactive ganglion cell axons C-dependent mechanisms (Goldman and Chiu, '84; Shafit- present in the lesioned retina, their fiber course was indistinguishable from that in the normal retina. We Zagardo et al.,'88). assume that these fibers are surviving axons for the followWe have not noticed an enhanced GFAP immunoreactivity within the retinal astrocytes, a feature which is known ing reasons. It is known that, in contrast to lower verteto occur in reactive astrocytes in the brain following stab brates, central nervous system (CNS) neurons of mammals wounds (Bignami and Dahl, '76; see also Reier, '86, for fail to regenerate their axons in response to injury. It has review). However, in our study the "injury" had not been been assumed that in the mammalian CNS myelin breakset into the retina itself, but in the optic nerve. We observed down products prohibit fiber regeneration, since mammathat the nerve fiber layer of the retina became considerably lian CNS neurons are capable of regrowing their processes thinner. Thus the thickness of this layer is not preserved by through pieces of transplanted peripheral nerves (So and astroglial processes forming a scar and filling all the space Aguayo, '85).In addition, it is known that CNS neurons are left by degenerating axons. It will be interesting to under- capable of sprouting and thus regenerate their processes stand why the stimulus inducing Muller cells to become when they are located in unmyelinated regions of the GFAP positive fails to enhance GFAP immunoreactivity in mammalian CNS, like the retina (Barron et al., '86; Eysel and Peichl, '85; McConnell and Berry, '82; Peichl and Eysel, astrocytes. In contrast to the transient GFAP labeling of Muller '86). It was shown that when a lesion was performed within cells, the staining intensity of the second intermediate the retina the regenerating nerve fibers took an unoriented filament protein present in Miiller cells, vimentin, seems random course; they did not run straight towards the optic not to be affected by optic nerve cut. However, during disc (Eysel and Peichl, '85; McConnell and Berry, '82). In long-term retinal detachment, the expression of vimentin our study, only very few randomly oriented, most likely by retinal Muller cells can well increase (Lewis et al., '89). regenerated, fibers were seen when retinal wholemounts Thus, retinal detachment and optic nerve cut may well lead were stained with neurofilament antibodies more than 2 years after optic nerve cut, and these fibers were confined to to different activation of Muller cell components. Microglial cells become vimentin positive in situ when the myelinated region of the rabbit's retina, the medullary activated by facial nerve axotomy (Graeber et al., '88). We rays. There is another argument in favor of the assumption have shown recently that, following optic nerve cut, microglial cells in the retina are activated. They begin to prolifer- that most ganglion cell fibers present after 2 years are ate and acquire higher enzymatic activity of their marker original axons. R.ecent studies have shown that rat oligodenenzyme nucleoside diphosphatase (Schnitzer and Scherer, drocytes and CNS myelin inhibit neurite outgrowth and '90). Nevertheless, we were not able to discover any vimen- fiber regeneration (Schwab and Caroni, '88). If this holds tin-labeled microglial cell at any survival time studied. true for the rabbit retinal myelin then we would not expect Thus, at least in our system, the observation reported by regenerating ganglion cell axons to elongate through (i.e., Graeber et al. ('88) could not be confirmed. However, we to prosper in) this myelinated region. What we observed, know that in vitro microglial cells are clearly vimentin however, was that the ganglion cell axons took a straight

6)

RETINAL NEUROGLIA AND GANGLION CELLS

Fig. 12. Electron micrograph of an optic nerve that had been transected intraorbitally 25 months before. The micrograph was taken close to the chiasm. The nerve consists of astroglial and oligodendro-

187

glial (oligo) nuclei, and numerous astroglial processes (asterisk) loaded with intermediate filaments. Four myelin-like structures are visible; one surrounds an axon-likefiber (Ax). Scale bar = 1 ym.

188

J. SCHERER AND J. SCHNITZER

Fig. 13. Semithin vertical sections through the medullary ray region. a: Lesioned retina, 25 months after nerve cut. b: Control retina. Both sections were taken about 5 mm temporal to the optic disc.

Numerous neuroglial cells are seen in the thinner nerve fiber layer of the lesion4 retina (a)but also in the control retina (b).Scale bar = 50 p,m.

course towards the optic disc which thus most likely survived for yet unknown reasons. It has been shown that growth factors like nerve growth factor or basic and acidic fibroblast growth factor promote the survival of rat retinal ganglion cells after nerve cut (Carmignotoet al., ’89; Severs et al., ’87). It remains to be shown whether growth factors are responsible for the survival of at least some ganglion cells after optic nerve cut. It might also be interesting to see whether 2 years after injury the surviving ganglion cells are in principle capable of elongating their axons if they were confronted with a suitable graft, like a piece of a peripheral nerve. It has been described that the closer the transection of an axon occurs to its perikaryon the faster is the degeneration of its soma (reviewed by Cole, ’68). We can only speculate whether oligodendrocytes or their myelin sheath may have a “trophic” (stabilizing) effect on axons, thus preventing them from rapid retrograde disintegration. We will discuss below that oligodendrocytes located in the nerve fiber layer of the “le~ioned”rabbit retina are somehow “activated” by the transection because they begin to

form aberrant myelin which covers their own plasma membrane.

Newroglial cells in the nerve fiber layer of the retina Astrocytes and oligodendrocytes were detectable in the nerve fiber layer after long term survival by means of immunocytochemistry and as revealed at the ultrastructural level. The total number of glial cells located in this layer remained rather constant. This is in accord with observations on Wallerian degeneration in the rat optic nerve (Vaughn et al., ’70). Since the nerve fiber layer shrinks following nerve transection, glial cells occupy a larger proportion of the space in this layer than normally. It is known that the number of astroglial cells is lower in thinner and higher in thicker nerve fiber layers (Bussow, 1980; Karschin et al., ’86a; Ogden, 1978). Since the “degenerated” retina has a thinner nerve fiber layer, one might expect a reduced number of astrocytes. This was, however, not observed in the mouse retina following nerve

):c

189

RETINAL NEUROGLIA AND GANGLION CELLS

L

6I

C

R

B A

0 P R

I I

A

t

L

I

I I

RIGHT (CONTROL )

LEFT ( L E S I O N E D )

A

B

C NFL

100 pm

Fig. 14. Camera lucida drawings of semithin vertical sections through the medullary rays. Retinal tissue was taken from the medullary ray region at various distances from the optic disc as indicated in the schematic drawing (top). A, B, and C were located 7, 5, and 2 mm temporal to the optic disc. Each round dot in the nerve fiber layer

represents a neuroglial cell nucleus. Note that they are more densely packed in the thinner nerve fiber layer of the lesioned retina. For clarity, only the positions of the nerve fiber layer (NFL) and outer nuclear layer (ONL) are indicated.

crush (Allcutt et al., '84a). In our study, the total number of neuroglial cells (astrocytes and oligodendrocytes)was rather constant. Oligodendrocytesrarely proliferate in response to injury (Skoff, '75). We therefore suggest, although it has to be proven, that the proportion of astrocytes to oligodendrocytes might not change. This is in accord with observations on glial cells in a degenerating adult rat optic nerve (Vaughn et al., '70). However, experiments in the cat retina where a degeneration of axons was caused by photoco-

agulator lesions have shown that the number of astrocytes is reduced in the axon-free sector (Karschin et al., '86b). It remains to be seen whether the type of lesion performed influences the survival of astrocytes in the nerve fiber layer. Several months after transection quite a few retinal oligodendrocytes were seen whose own plasma membrane was covered with a myelin sheaths. This has so far only been described in a late stage of Wallerian degeneration of

190

J. SCHERER AND J. SCHNITZER

TABLE 1. Comparison of Neuroglial Cell Numbers in the Nerve Fiber Layer of a “Lesioned” Retina 2 Years After Optic Nerve Transection and of a Control (Right)Retina’ Area examined A B C

Lesioned retina

Control retina

32 82 231

31

79 200

To obtain these numbers, neuroglid cell nuclei were counted in Nissl-stained semithin sections. Sections from three respective regions in the lesioned (left) and control (right) retinawere evaluated. Areas A, B, and C were located in the medullaryrays as indicatedin the scheme in Figure 13. The values given represent the number of neuroglial cells found in the nerve fiber layer within 1mm of length.

optic nerve of rats (Vaughn and Pease, ’70) and also in the spinal cord (Bignami and Ralston 111, ’68, ’69). This means that after disappearance of about 90% of the ganglion cell axons, oligodendrocytes form aberrant myelin around

Fig. 15. Electron micrograph of the medullary ray region of a lesioned retina 9 months after optic nerve transection. Many healthylookingmyelinated axons (Ax) are present. An axon-like fiber (indicated

“false” membranes. The signal that triggers oligodendrocytes to form aberrant myelin remains to be elucidated.

ACKNOWLEDGMENTS We thank Dr. S. Thanos and Dr. N. Brecha for advice in transecting the optic nerve of rabbits, Dr. B. Ranscht for the gift of galactocerebroside antibodies, Dr. L. Peichl for performing stereotactic injections of HRP into the superior colliculus of rabbits, Dr. H. Wolburg for helpful discussion of electron microscopic observations, Dr. J. Nelson for linguistic advice, and Prof. Wassle for continuous encouragement and support. The excellent technical assistance of W. Hofer, G.-N. Nam, and C. Ziegler is gratefully acknowledged. Some of this work was carried out as part of the Ph.D. thesis project of J. Scherer.

by an asterisk) is seen that is surrounded by an oligodendroglial process. An oligodendroglia cell body (oligo) is covered by an aberrant myelin sheath. Scale bar = 1p,m.

RETINAL NEUROGLIA AND GANGLION CELLS

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