GLIA 3:33-42 (1990)

Expression of Glial Fibrillary Acidic Protein (GFAP) In Goldfish Optic Nerve Following Injury C.A. STAFFORD, S.A.S. SHEHAB, S.N. NONA, AND J.R. CRONLY-DILLON Developmental Neurobiology Laboratory, Department of Optometry and Vision Sciences, UMIST, Manchester, M60 lQD,England

KEY WORDS

Regeneration, Reactive astrocytes, Immunoblotting, Immunohistochemistry

ABSTRACT

By using an antibody to goldfish glial fibrillary acidic protein (GFAP), the reaction of goldfish optic nerve to injury has been studied by immunoblotting and immunohistochemical methods. Goldfish optic nerve, which normally lacks GFAP immunoreactivity (Nona et al.: Glia, 2:189-200, 1989), expresses GFAP following injury. This immunoreactivity, which is observed as early as 10 days after crush and which is still evident at 30 days after crush, all but disappears by 150 days after crush. Since it is well established that functional restoration of synaptic connections and the recovery of vision takes place in goldfish following optic nerve injury, our results indicate that reactive astrocytes do not represent an impediment to regeneration in goldfish visual system.

INTRODUCTION One of the areas of neuroscience research that has attracted a great deal of attention is that of central nervous system (CNS) regeneration (Kao et al., 1983; Kiernan, 1979; Windle, 1955). In human and other higher vertebrates, following an injury to brain, spinal cord, or optic nerve there is an almost complete failure by any new fibres to cross the injured zone to make functional connections with their target. In many lower vertebrates, on the other hand, there is a considerable capacity for functional regeneration by the damaged CNS (Attardi and Sperry, 1963; Gaze, 1959,1960). Of the many reasons put forward to explain the lack of regeneration in CNS of higher vertebrates, that of scar formation at the site of injury is often considered (Reier et al., 1983). Such a scar consists of a matrix of enlarged astrocyte cell bodies and interweaving cytoplasmic processes. In filling the spaces left vacant by the degenerating neural tissue, these so-called reactive astrocytes appear to present an obstacle to any neuronal growth in higher vertebrates (Berry, 1979; Reier et al., 1983; Wujek and Reier, 1984). Although astrocytes in amphibia (Reier, 1979; Reier et al., 1983; Stensaas and Feringa, 1977) and fish (Wolburg, 1981) also appear to undergo hypertrophy and proliferation followinginjury, 01990 Wiley-Liss, Inc.

they somehow appear to provide a more agreeable environment for successful axonal regeneration than their counterparts in higher vertebrates. Goldfish visual pathway has long been considered a model system for studies that are pertinent to successful functional regeneration (see, for example, Edds et al., 1979). In an attempt to unravel the molecular events associated with this regeneration (Grafstein and Murray, 1969), particular attention has been focused on the role of certain axonally transported proteins, designated “growth-associated proteins,” which are transported within the fast phase of axonal transport and which show a marked increase in synthesis during regeneration (Benowitz and Lewis, 1983; Heacock and Agranoff, 1982; Perrone-Bizzozeroand Benowitz, 1987). In addition, the role of certain intermediate filament proteins has also been investigated (Perry et al., 1985; Quitschke and Schechter, 1983a). These proteins are either transported along the regenerating axons within the slow phase of axonal transport or are present within the environment surrounding these axons (Deaton and Received June 26,1989; accepted September 5,1989. Address reprint requests to C.A. Stafford, Developmental Neurobiology Laboratory, Department of Optometry and Vision Science, UMIST, Manchester,M60 1QD England.

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Freeman, 1983; Jones et al., 1986; Perry et al., 1987; Quitschke and Schechter, 1984) and have been considered to play a role in axonal growth and plasticity of the system. To date, the most studied of non-neuronal proteins from goldfish visual pathway have been those termed ON3 and ON4, of molecular weight 58 kDa (Quitschke and Schechter, 1983a, 1984). Although these so-called glial proteins are believed to be present in astrocytes, they do not cross react with antibodies to the conventional astrocyte proteins glial fibrillary acidic protein (GFAP) and vimentin (Quitschke et al., 1985; see also, Maggs and Scholes, 1986). We have recently been successful in raising an antigoldfish GFAP which recognises, in normal goldfish, ependymal glia population in tectum, long processes in tract, and Muller fibres in retina, but, surprisingly, no GFAP immunoreactivity is observed in optic nerve (Nona et al., 1989). The present study utilises this antibody to monitor the effects of optic nerve injury on the expression of GFAP-positive astrocytes in goldfish optic nerve. The results are discussed in terms of the role of astrocytes during optic nerve regeneration. MATERIALS AND METHODS Animals Common goldfish (Carrasius auratus), 5-7 cm in length, were obtained from a local pet shop and were kept in gravel-bottomed glass tanks containing aerated tap water at 19-21°C. Retrobulbular crush of optic nerve was carried out in anaesthetised animals (0.4% MS 222; BDH). Connective and fatty tissues were first removed from around the eye, and the nerve was grasped with fine watchmakers' forceps, at about 1 mm from the eye ball, and forcibly squeezed for about 5 sec. Either the left optic nerve was crushed with the right nerve serving as a control or both optic nerves were crushed, in which case the nerve from an unoperated animal was used as a control. At predetermined times following optic nerve crush, tissue was removed for processing by the methods described below.

Gel Electrophoresis Preparation of samples for electrophoresis was carried out according to the method of Laemmli (Laemmli, 1970). Comparison of the composition of goldfish proteins from normal and crushed optic nerves was carried out on the Phast System electrophoresis (Pharmacia), using 10-15% gradient SDS-polyacrylamidegels.

Immunoblotting Proteins from the above preparations, after separation on one-dimensional SDS-polyacrylamidegel, were transferred t o nitrocellulose according to an established procedure (Towbin et al., 1979). Blots were blocked in 3%normal swine serum in a buffer consisting of 20 mM Tris (pH 7.3) and 150 mM NaCl for 1h at 37°C. Incubation with antigoldfish GFAF' (1:200) was carried out at room temperature overnight. Blots were thoroughly washed with the above buffer and incubated for 1h at room temperature with horseradish peroxidase (HRP)conjugated antirabbit IgG ( 1:200; Sigma). Thoroughly washed blots were then visualised by development in the above buffer containing 3,3'-diaminobenzidine (0.05%) to which was added hydrogen peroxide (0.003%).The reaction was terminated with distilled water, and the blots were photographed immediately.

Immunohistochemistry

Goldfish optic nerve, often with tract, was dissected out and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) for 4 h. Tissue was washed and stored in 15% sucrose in phosphate-buffered saline (PBS; pH 7.3) overnight, frozen in liquid nitrogen, and cut on a cryostat (-20°C). Sections (20 pm) were picked up on a gelatin-coated slides, dried at room temperature for 1h, and then washed thoroughly with 0.1 M PBS (pH 7.3) followed by PBS containing 0.2% Triton X-100. Sections were incubated with antigoldfish GFAP (1:400)at 4°C in a humid chamber overnight. This w'as followed by successive Preparation of Tissue Homogenates hourly treatments at room temperature with swine Two different protein preparations were used in this antirabbit (150; Dako) and peroxidase-antiperoxidase study. First, an intermediate filament fraction was (1:lOO; Dako). Sections were reacted for 10 min with obtained from normal optic nerves of 10 goldfish, as 3,3'-diaminobenzidine (0.0547)in PBS (pH 7.3) to which described previously (Nona et al., 1989),according to an was added hydrogen peroxide (0.003%) before being established procedure (Chiu et al., 1981). Second, for dehydrated through graded alcohol, cleared with xyall nerve-crushed animals, a total protein preparation lene, and cover-slipped for observation with a Leitz was obtained by homogenising optic nerves of one or Diaplan light microscope. Alternatively, sections incutwo goldfish in 0.4% sodium dodecyl sulphate (SDS) or bated with primary antibody were then incubated for directly in sample buffer containing 1% SDS. The 1h at room temperature with tetramethyl (TRITC) homogenates were centrifuged at 10,OOOg for 15 min rhodamine-conjugated goat antirabbit IgG (150; at 4"C, and the pellet was discarded. The superna- Sigma), washed well with PBS (pH 7.3), covered with tants, which had a protein concentration in the range glycerine in PBS (3:1), and viewed through a Leitz of 4-5mg/ml (Bradford, 19761, were kept at -23°C Diaplan microscope equipped with epifluorescence illuuntil use. mination and filters for fluorescence.

GFAP IN INJURED GOLDFISH OPTIC NERVE

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nerve by the use of cobalt chloride and nickel ammonium sulphate reagents gave unacceptably high backThe cytoskeletal fraction from noncrushed optic nerve ground, with no obvious improvement in the band intenwas used t o ascertain whether GFAP protein, as deter- sity (data not shown). mined by electrophoresis and immunoblotting, was present in normal optic nerve of goldfish. For reasons of economy, an optic nerve total protein preparation Operated goldfish rather than a cytoskeletal preparation was used for the Coomassie blue-stained one-dimensional gels of total corresponding GFAP studies in nerve-crushed goldfish. protein fractions from goldfish optic nerve at 10,15,21, 25,30, and 150 days after crush revealed, as expected, a much greater distribution of protein bands than that Biochemical Localisation of GFAP in Goldfish observed for the cytoskeletal fraction (Fig. 1A). The Optic Nerve major protein bands were now in the region of 55 kDa (tubulin) and 43 kDa (actin), with the cytoskeletal proNormal goldfish teins, including any GFAP, being considerably diluted Coomassie blue-stained one-dimensional gel of inter- out (Fig. 1A).Nevertheless, except for the fraction from mediate filament fraction from normal optic nerves 150 days after crush, the presence of GFAP was conshowed a distribution of protein bands primarily in the firmed by immunoblot using antigoldfish GFAP for all molecular weight range of 45-145 kDa (Fig. lA), as of the other optic nerve crush fractions (Fig. lB,C). This previously reported (Nona et al., 1989; Quitschke and result not only demonstrates the specificity of the antiSchechter, 1984; Quitschke et al., 1980). Two major body but also adds weight to the above finding that protein bands were apparent at 48 kDa and 58 kDa, GFAP in normal optic nerve is indeed present in only while the presence of a 51 kDa band, corresponding to trace amounts, if at all. In general, the staining of the GFAP, was not readily recognisable. This latter obser- GFAP band in immunoblots appeared t o be of approxivation was confirmed by immunoblot technique, using mately equal intensity for all of the optic nerve fractions antigoldfish GFAP, which revealed the virtual absence except for that from 21 days after crush, which showed a of this protein even after prolonged immunoblotting stronger staining (Fig. 1C).However, this stain was still (Fig. 1B).Attempts to augment the blot of normal optic considerably weaker than that for the corresponding RESULTS

Fig. 1. A Coomassie blue-stained one-dimensional 10-15% gradient gel of SDS-soluble total protein fraction from goldfish optic nerve at 150 days after crush (lane 1)and at 21 days after crush (lane 2) and from intermediate filament fraction from normal goldfish optic nerve (lane 3). Lane 3 shows protein bands at a: 145 kDa, b: 80 kDa, c: 58 kDa, and d 48 kDa. Lane 2 shows additional protein bands at e: 55 kDa and E 43 kDa, corresponding to tubulin and actin, respectively, with a similar distribution in lane 1 (twice loading of lane 2). Lane 4 shows purified GFAP band from goldfish brain. Lane 5 shows molecu-

lar weight standards (Sigma): phosphorylase B (97.4 kDa), albumin bovine (66 kDa), ovalbumin (45 m a ) , and glyceraldehyde-3-phosphate dehydrogenase (36 kDa). B: Immunoblot corresponding to gel A, showing the specificity of antigoldfish GFAP (lane 4). GFAP immunoreactivity is virtually absent in protein extracts from normal optic nerve (lane 3) and from optic nerve at 150 days after crush (lane l ) , but is clearly evident in optic nerve at 21 days after crush (lane 2). C: Immunoblot of total protein extracts from optic nerve at 10 (lane l ) , 15 (lane 2), 21 (lane 3), 25 (lane 4), and 30 (lane 5) days after crush.

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Longitudinal sections of optic nerve from all of the operated goldfish, except from those 150 days after crush, showed very clearly positive immunoreactivity with antigoldfish GFAP, thus confirming the above biochemical results. GFAP immunoreactivity was ob-

served proximal and distal t o the site of injury, with the greatest staining being in the areas directly adjacent to the site of injury, as shown, for example, for the 21 day postcrush material (Fig. 3). The pattern of staining consisted of an elaborate network of short processes traversing the whole of the nerve. Upon closer inspection, this network was seen to be made up of a number of fascicles, separated from each other by connective tissue. These fascicles, in turn, were made up of numerous bundles that showed GFAP :.mmunoreactivity at their rims (Fig. 4).The origin of these GFAP processes and, therefore, their cell bodies could not be discerned. In contrast to this positive GFAP immunoreactivity observed in optic nerve during the early stages of regeneration, optic nerve at 150 days after crush contained only very few GFAP-positive processes (Fig. 51, with the rest of the nerve resembling that from a normal goldfish. The pattern of staining in the tract of the injured goldfish remained strongly GFAP positive, similar to that from the normal goldfish (data not shown). Interestingly, throughout t he regeneration period the injured area, which was readily discernible and approximately 0.3-0.5 mm in width. remained almost entirely free of any GFAP immunoreactivity, with only the occassional GFAP-positive process being observed within this region (Fig. 31, even though the adjacent areas were strongly GFAP positive. This lack of immunostaining in the injured area was consistently observed in all of the operated goldfish by immunofluorescence as well as by PAP staining. This lack of

Fig. 2. Longitudinal section showing optic nerve (N) and tract (T) from normal goldfish stained with antigoldfish GFAP (TRITC).In optic nerve, hardly any GFAP-positive structures are evident, although weak, nonspecific staining is observed (small arrow). In tract, GFAPpositive long processes (large arrow) are observed emanating from the

ventricle (V) and then running parallel within the tract. Also observed in the tract are GFAP-positive radial tufts (black arrowhead) associated with glia limitans surface (gl). Note the impressive immunoreactivity demarcation (white arrowhead) between optic nerve and tract, just beyond the chiasm. Bar = 100 pin,

tract fractions (data not shown). It is worth noting that normal tract, unlike normal optic nerve (Fig. 21, has been shown to be strongly GFAP-positive by biochemical and histochemical methods (Nona et al., 1989). Jmmunohistochemical Localisation of GFAP in Goldfish Optic Nerve Normal goldfish As previously described (Nona et al., 19891,longitudinal sections of normal optic nerve showed hardly any GFAP immunoreactivity with antigoldfish GFAP (Fig. 2). By contrast, optic tract beyond the chiasm stained heavily with antigoldfish GFAP; coarse processes running parallel to the direction of the nerve fibres were observed throughout the tract. A considerable number of these processes appear to emanate from the heavily stained ventricle region, while a smaller proportion of the processes were in the form of tufts adjacent to the glia limitans surface (Fig. 2). Operated goldfish

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GFAP IN INJURED GOLDFISH OPTIC NERVE

Fig. 3. Longitudinal section showing optic nerve at 21 days after crush stained with antigoldfish GFAP

(TRITC).A network of GFAP-positive short processes is clearly evident both proximal (P)and distal (D) to the site of crush (C). In the crushed zone, very few such processes are evident (arrow). Bar

=

100 pm.

Fig. 4. A higher magnification of a section from optic nerve at 21 days after crush, immediately distal to the site of crush (C), stained with antigoldfish GFAP (PAP), It shows large fascicles (F) separated by connective tissue. These fascicles are made up of a number of bundles (b) that show GFAP immunoreactivity at their rims. Bar = 50 pm.

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Fig. 5 . LonBtudinal section showing optic nerve a t 150 days after crush stained with antigoldfish GFAP (TRITC).Very few GFAP-positiveprocesses are observed (arrow).The crushed zone appears;to have healed completely (arrowheads). Bar = 100 pm.

immunostaining at the injured area was also observed structural studies. Significantly, in the abovementioned teleost examples, ilstrocytes also show GFAP with antihuman GFAP (data not shown). expression in the normal unregenerated tissue. Furthermore, attempts to study the expression of GFAP in DISCUSSION the visual system of normal (Dahl and Bignami, 1973; Of the many morphological and cellular events ac- Quitschke et al., 1985) and injured (Bignami and Dahl, companying scar formation in injured central nervous 1976) goldfish have been unsuccessful. Lack of specific system, that of an increased density of 10 nm filaments anti-GFAP has been one of the major obstacles to such within the astrocyte processes is the most predominant studies (Bignami and Dahl, 1976). Such a difficulty was (Reier et al., 1983). These filaments are considered to recently overcome following our isolation of an antibody be, primarily, polymers of astrocyte-specific GFAP, as to goldfish GFAP that recognises GFAP-positive they react heavily with anti-GFAP (Dahl et al., 1986; ependymal glia in normal goldfish tectum, long proEng, 1980). Indeed, filaments isolated from severely cesses in tract, and Muller fibres in retina; but, surprisgliosed human tissue provided the first source of GFAP ingly, no GFAP-positive structures were observed in (Bignami et al., 1980; Eng et al., 1971; Goldman et al., optic nerve (Nona et al., 1989 1. This antibody, therefore, appears to be ideally suited for the study of any GFAP 1978). In an attempt to understand more fully the role of expression in goldfish optic nerve during regeneration. glial scarring in CNS regeneration, the response of Throughout this study, use was made of two methods for astrocytes to injury has been studied in a number of the monitoring of GFAP expression: immunoblotting lower vertebrates. In transected spinal cord of goldfish, and immunohistochemistry. These methods were utifor example, regeneration takes place (Bernstein and lised primarily for the detection rather than estimation Bernstein, 1967) despite the appearance of fibrous glio- of GFAP. Both methods performed well, and the results sis, as judged by immunoreactivity to antihuman GFAP were in general agreement. Of the two methods, howin proximal and distal stumps (Bignami et al., 1974). In ever, immunoblotting appears to be more suitable for Sternarchus fish, transection of spinal cord also leads to the detection of small changes in GFAP (Fig. 1C) and an increase in GFAP immunoreactivity at the transition would, therefore, be a better candidate for any quantizone between regenerated and unregenerated sections tative studies. In the present work, the normally GFAP-negative of cord (Anderson et al., 1984). In regenerating optic nerve of newt (Stensaas and Feringa, 1977) and Xeno- goldfish optic nerve (Nona et al., 1989) shows GFAP pus (Reier et al., 1983),reactive astrocytes are the major reactivity following optic nerve crush, on the basis of component of the cranial stump, according to ultra- immunoblotting (Fig. lB,C) and immunohistochemical

GFAP IN INJURED GOLDFISH OPTIC NERVE

(Fig. 3) results. This expression of GFAP in optic nerve is observed as early as 10 days after injury and is still evident at 30 days after injury. Throughout this period, a network of GFAP-positive processes is observed both proximal and distal to the injured zone. The injured region, on the other hand, remains almost totally devoid of any GFAP immunoreactivity (Figs. 3, 4). This situation of GFAP expression in regenerating goldfish optic nerve appears to resemble that observed in regenerating goldfish spinal cord, where fibrous gliosis was noted in the proximal and distal stumps but not in the injured area (Bignami et al., 1974). These results from goldfish studies, however, appear to be at odds with those from the regenerating spinal cord in Sternarchus fish, where positive- GFAP staining, as detected by antihuman GFAP, was observed at the transition zone between regenerated and unregenerated cord (Anderson et al., 1984). Since in the present work GFAP reactivity in the crushed area was not detected either by antigoldfish GFAP (Fig. 3) or by antihuman GFAP (data not shown), one may agree with the suggestion that the mechanism for spinal cord regeneration in Sternarchus fish is different from that for goldfish (Anderson et al., 1984). Yet another interesting observation from the present study is the fact that there was an almost total loss of GFAP immunoreactivity in goldfish optic nerve at 150 days after injury (Fig. 5). At this stage, and perhaps even earlier, optic nerve axons would be expected to have invaded their target tectum and to have re-established effective synapses, with a high degree of retinotopic order (Schmidt and Edwards, 1983), and there would be recovery of vision (Wolburg and Kastner, 1984). No comparable decline in GFAP immunoreactivity has been reported in the other regenerating systems even though, as in the case of Sternarchus fish, such a study was conducted as late as 4 months after spinal injury (Anderson et al., 1984),when regeneration would be almost complete (Anderson et al., 1983). How can the results from the present study, then, be related to what is already known about some of the events taking place during optic nerve regeneration in goldfish? Studies using electron microscopic technique indicate that regeneration in goldfish optic nerve is accompanied by glial proliferation, including astrocyte proliferation, as early as 4 days after nerve injury. This proliferation remains at a high level until around 57 days after injury, after which it begins t o decline (Wolburg, 1981; Wolburg and Kastner, 1984). On the other hand, during optic tract regeneration, the number of oligodendrocytes, but not of astrocytes, increases, although there is an increase in the number of astrocyte processes (Giulian and Iwanij, 1985; Giulian et al., 1985). In the present work we are unable to establish whether the induction of GFAP expression in regenerating optic nerve is due to astrocyte proliferation, since no GFAP-positive astrocyte cell bodies could be discerned before or after nerve injury. Nevertheless, if the GFAP expression during regeneration is due to astrocyte proliferation, then the crushed zone, which, according to Wolburg (19811,contains proliferating astrocytes,

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should show strong GFAP immunoreactivity; this, however, was not observed (Fig. 3). We believe that this discrepancy can be explained thus: The fact that Wolburg (1981) considers the crushed zone in his experiments to be ca. 1mm wide (compared with less than 0.5 mm in our studies) suggests that he has included in his observations the distal part of the nerve between the crushed zone and the chiasm. In such a situation, the induction of GFAP reactivity in the distal (and proximal) parts of the optic nerve observed in our studies may well be due in part to astrocyte proliferation, as Wolburg (1981)suggests. Additionally, however, this induction of GFAP expression may also be attributed to growth of astrocyte processes (Giulian et al., 19851, coupled with an increase in the translation of the pre-existing GFAP mRNA or de novo transcription of GFAP mRNA (Eng, 1987).These events appear not to be taking place in the crushed zone, but why? If, as has been proposed, astrocytes play a guidance role during optic nerve regeneration in goldfish (Wolburg and Kastner, 1984) and in newt (Turner and Singer, 1974), then it becomes difficult to explain the observation from the present study as to why GFAPpositive astrocytes are steering clear of the injured area, where they may be needed for guidance. One possible explanation may be derived from the related studies of optic nerve regeneration in newt, where the injured zone, followingfreeze-crush, has been shown to lack any viable cellularity, apart from proliferating macrophages, as late as 14 days after injury (Stensaas and Feringa, 1977).A similar situation may well be prevailing in crushed zone of goldfish optic nerve, where, despite considerable cellular proliferation at the site of injury as early as 4 days after crush (manuscript in preparation), none of it appears to be related to astrocytes proliferation, as shown by lack of GFAP immunoreactivity. However, there the similarity between the two systems ends, since in the case of newt longitudinally oriented astrocyte processes are observed crossing the crushed zone from the adjacent retinal stump at 10 days after nerve crush, while in goldfish this is not observed even at 30 days after nerve crush, when nerve fibres would be expected not only to have crossed the crushed zone but also to have reached the tract (Lowenger and Levine, 1988). It seems that if the new fibres are closely surrounded by glial cells, as previously reported (Murray, 19821, then these glial cells do not express GFAP in the crushed zone. To date, we have not found it possible to determine immunohistochemically the nature of any cells in the crushed area, although a number of antibodies to microglia and macrophages have been tried. However, it is important to continue this line of investigation by the use of electron microscopic technique. At 150 days after crush, GFAP immunoreactivity in proximal and distal parts of regenerating goldfish optic nerve is almost completely lost. At this stage of regeneration, it may be assumed, retraction of astrocyte processes is taking place, and that optic nerve is returning to its normal architecture. This expression and de-

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expressionof GFAP in regenerating goldfish optic nerve is yet to be demonstrated in other lower vertebrates, among which amphibia are most suitable because of the lack of conventional GFAP in their optic nerve (Szaro and Gainer, 1988; however, see Naujoks-Manteuffel and Roth, 1989). It is worth pointing out that throughout regeneration the tract, unlike optic nerve, fails to show any noticeable increase in GFAP expression (data not shown). It is possible that some increase in GFAP reactivity does take place in the tract during regeneration, but is not visualised very clearly because of the presence of strong stain in the normal tract. However, it is equally possible that tract may contain astrocytes that are reacting differently t o those in optic nerve. Indeed, from a morphological standpoint, astrocytes in injured optic nerve, excluding the crushed area, appear as a compact network (Fig. 31, and this pattern contrasts with the more open structure of glial processes observed in the tract (Fig. 2). The functional significance of this astrocytic diversity is yet to be unravelled, although it has been suggested that the compactness of optic nerve astrocytes may serve t o shepherd and support the developing (Maggs and Scholes, 1986)and regenerating (Lowenger and Levine, 1988)fibres, while the more loosely organised astrocytes of the tract are involved in the correct sorting out of the fibres as they cross the chiasm. It is interesting that although astrocytes from normal goldfish optic nerve do not express GFAP (Dahl and Bignami, 1973; Dahl et al., 1985; Nona et al., 1989; Quitschke et al., 1985),they have been shown t o express other non-neuronal markers in the range of 58 kDa (Quitschke and Schechter, 1984).These “glialproteins,” designated ON3 and ON4, do not show any crossreactivity with antibodies to GFAP or vimentin (Quitschke et al., 19851, the two proteins most commonly coexpressed in adult rat astrocytes such as Bergmann, Muller, and radial glia (Pixley and De Vellis, 1984; Schnitzer et al., 19811, as well as in optic nerve astrocytes (Dahl, 1981). Furthermore, in response to optic nerve injury, there is little change in the concentration of ON3 and ON4, as observed biochemically (Quitschke and Schechter, 198313) and histochemically (Jones et al., 19861, a result that was interpreted as evidencefor lack of any appreciable gliosis in regenerating optic nerve of goldfish (Jones et al., 1986). Quite clearly this conclusion is not substantiated by the results from the present study, which demonstrate that gliosis, as defined by an induction of GFAP expression (probably associated with an increase in the number of cells and processes) is indeed taking place in this system. Nevertheless, it seems curious that it is the normally absent GFAP, an astrocytes protein often associated with the state of maturation in higher vertebrates (Dahl, 1981; Harry et al., 1985), that is expressed in regenerating goldfish optic nerve. Interestingly, it has recently been suggested that these ON3 and ON4 proteins in goldfish and their equivalent in cichlid fish (Maggs and Scholes, 1986) are probably of cytokeratin origin (Mark1 and Franke, 1988; Rungger-Brandle et al., 1989bbased on studies of rainbow trout and

amphibia, respectively. In this context it is worth noting that astrocytes in normal rat optic nerve coexpress GFAP and vimentin. Following Wallerian degeneration, reactive astrocytes accumulate not only GFAP but also vimentin in the form of 10 nm filaments, resulting in fibrous gliosis (Dahl et al., 1981a,b). Despite these differences in the cytoskeletal composition of optic nerve astrocytes, results from the present study show that astrocytes in regenerating goldfish optic nerve behave in a manner similar to that in injured rat optic nerve: That is, hoth systems accumulate GFAP. The functional significance of this increase in intermediate filaments is not, known, but it appears to be a typical astroglial reaction to injury (Eng, 1988; Nieto-Sampedro et al., 1985)in both degenerating and regenerating systems. Indeed, results from the present study also suggest that the accumulation of GFAP in reactive astrocytes following nerve injury and the apparent peak at 21 days after injury (Fig. 1C) do not correspond to any specific stages of early fibre regeneration (Lowenger and Levine, 1988).These reactive astrocytes, however, may be regulating the extracellular constituents, such as potass urn ions, nutrients, and trophic factors (Eng, 1988), as well as participating in the removal of myelin and neuronal debris from the injured area (Wolburg, 1981). This latter idea receives some support from our recent unpublished observation that, 10 days after goldfish eye removal, optic nerve shows an accumulation of GFAP, which then all but disappears by 70 days follofiing injury. With this in mind, one may conclude from the results of the present study that, despite the accumulation of GFAP and perhaps because of it, regene7ation still proceeds successfully in goldfish visual system. Perhaps lack of regeneration is due to the presence of reactive astrocytes that express a particular type of intermediate filament protein. Such an idea seems plausible, especially in light of the recent finding (Rungger-Brandle et al., 1989) that astrocytes in amphibian optic nerve express cytokeratin rather than vimentin. We are now embarking on studies of such ideas. ACKNOWLEDGMENTS We thank the Wellcome Trust for financial support.

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site of injury in the optic nerve of newt Triturus pyrrhogaster. Cell Wolburg, H. (1981) Axonal transport, degeneration and regeneration Tissue Res., 179501-516. in the visual system of the goldfish. Adu. Anat. Embryol. Cell Biol., Szaro, B.G., and Gainer, H. (1988) Immunocytochemical identification 67:l-94. of non-neuronal intermediate filament proteins in the developing Wolburg, H. and Kastner, R. (1984) Astroglial-axonal interrelationXenopus laevis nervous system. Dev. Brain Res., 43:207-224. ship during regeneration of the optic nerve in goldfish. A freeze fracture study. J. Hzrnforsch., 25: 493-504. Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide to nitrocellulose sheets: Proce- Wujek, J.R. and Reier, P.J. (1984) Astrocytic membrane morphology: Differences between mammalian and amphibian astrocytes after dure and applications. Proc. Natl. Acad. Sci. U.S.A., 76:43504354. axotomy. J . Comp. Neural., 222:607-619. Turner, J.E., and Singer, M. (1974) The ultrastructure of regeneration in the severed newt optic nerve. J. Exp. Zool., 190:249-268. Windle, W.F. (1955)Regeneration in Central Nervous System. Thomas, Springfield, Illinois.