THE JOURNAL OF COMPARATIVE NEUROLOGY 305582-590 (1991)

Expression of Glial Fibrillary Acidic Protein by Muller Cells in rd Chick Retina SUSAN L. SEMPLE-ROWLAND Department of Neuroscience, University of Florida College of Medicine, Gainesville, Florida 32610-0244

ABSTRACT Accumulation of glial fibrillary acidic protein (GFAP) in Muller cells has been observed in retinas of several mammalian species secondary to genetically induced degeneration and neuronal injury. In the present series of experiments, I have examined the effects of the rd (retinal degeneration) mutation on the expression of GFAP in retinas of chicks homozygous for the mutation (rdlrd)prior to and following the onset of photoreceptor degeneration, which first appears approximately 7 days posthatch (7dph). Carrier ( + / r d ) and wild-type (+/+I retinas served as controls. Retinas taken from 1, 7,21, and 33 dph rdlrd, +/rd, and +/+ chicks were analyzed for the presence of GFAP by immunocytochemical and SDS-PAGEWestern blot techniques. The following immunocytochemical observations were made: (1) GFAP immunostaining was limited to and located throughout the Muller cells. (2) The intensity of GFAP immunostaining increased with age in all three retina types in tissue sections, as well as on immunoblots. (3) The distribution of GFAP staining within rdlrd Muller cells following the onset of degeneration was slightly different from that observed in +/+ and +/rd retinas and was distinguished by increased staining of the cell bodies and the cell processes forming the outer limiting membrane. The results of these experiments show that Muller cells in chick retina contain GFAP. In addition, they suggest that, in contrast to Muller cells in degenerating mammalian retina, Muller cells in rd chick retina do not accumulate large amounts of GFAP in response to degeneration. Key words: vimentin, cerebellum, intermediate filaments, retinal degeneration, GFAP

Since the early 1980s, several immunohistochemical studies have been conducted to examine the distribution of intermediate filaments in retina. Glial fibrillary acidic protein (GFAP), an intermediate filament component of astrocytes in the central nervous system (Eng et al., '71; Bignami et al., '72; Eng, '82, '851, has been the focus of several of these studies. Mammalian retina contains two types of neuroglia of neuroectodermal origin, Miiller cells and astrocytes (Cajal, '73; Rasmussen, K.-E., '75; Bussow, '80; Reichenbach and Wohlrab, '86; reviewed in Schnitzer, '88). Immunohistochemical analyses of normal mammalian retina have shown that astrocytes consistently stain with antibodies generated against GFAP (Shaw and Weber, '84; Schnitzer, '85; Stone and Dreher, '87; reviewed in Schnitzer, '88). Data concerning GFAF' immunoreactivity in Muller cells of these retinas are much less clear. Despite disagreement about the presence of GFAP in Muller cells of normal mammalian retina (see Discussion), accumulation of GFAP in these cells following retinal injury or degeneration has been consistently observed (Bignami and Dahl, '79; EisenO

1991 WILEY-LISS, INC.

feld et al., '84; Molnar et al., '84; Shaw and Weber, '84; Karschin et al., '86; Kivela et al., '86; Eisenfeld et al., '87; Erickson et al., '87; Ekstrom et al., '88; Lewis et al., '89; Sarthy and Fu, '89). Very few studies have been conducted to examine the distribution of GFAP in normal nonmammalian retina. Miiller cells have been reported to be GFAP immunoreactive in adult goldfish (Bignami, '84; Linser et al., '85; Jones and Schechter, '87; Nona et al., '89) and Xenopus retina (Szaro and Gainer, '88),but not in chicken retina (Lemmon and Reiser, '83). In this study, immunohistochemical and SDS-PAGE western blot techniques have been used to examine the effects of the retinal degeneration (rd) mutation on GFAP expression in chick retina. The rd chick possesses an autosomal recessive mutation that results in behavioral Accepted December 3,1990. Address reprint requests to Dr. Susan L. Semple-Rowland, University of Florida, College of Medicine, Department of Neuroscience, Box 3-244 JHMHC, Gainesville, FL 32610-0244.

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and electrophysiological blindness at hatch (Ulshafer et al., '84). The first signs of retinal degeneration begin to appear approximately 7-10 days posthatch (dph) and are restricted primarily to the photoreceptor cells. By 30 dph, a distinct retinal lesion is often seen in the posterior fundus just superior to the optic nerve head (Ulshafer and Allen, '85a,b). Based on reports that GFAP expression in Muller cells of mammals possessing hereditary retinal degenerations increases with the onset of degenerative changes (Drager and Edwards, '83; Eisenfeld et al., '84; Molnar et al., '84; Karschin et al., '86; Kivela et al., '86; Erickson et al., '87; Ekstrom et al., '88; Lewis et al., '89; Sarthy and Fu, '891, it was hypothesized that the Muller cells in rd chick retina would show similar increases in GFAP following the onset of photoreceptor degeneration. The results of the present immunohistochemical experiments conducted to test this hypothesis were somewhat unexpected and show (1)that increased expression of GFAP in Muller cells occurs during normal retinal development in chicken and (2) that retinal degeneration induced by the rd mutation is not accompanied by a significant increase in GFAP expression in the Muller cells.

minute washes in PBS, the sections were incubated for 1 hour in biotinylated horse antimouse IgG diluted 1 2 0 0 with PBS. The biotinylated antimouse IgG was preabsorbed with an equal volume of normal chick serum prior to dilution with PBS. Following incubation with the biotinylated secondary antibody, the sections were washed (3 x 10 min in PBS), and then incubated for 1hour in ABC reagent (Avidin DH: biotinylated horseradish peroxidase H complex).After buffer rinses, peroxidase activity was visualized by incubating the sections in 3,3'-diaminobenzidine tetrahydrochloride (0.03% w/v) and hydrogen peroxide (0.0006%) in 0.1 M Tris-HC1 buffer (pH 7.2) for 10 minutes. Incubated sections were rinsed in buffer, collected on chrome alum gelatin-coated slides, and allowed to air dry. Dried sections were dehydrated in an alcohol series, cleared in xylene, and coverslipped with Permount mounting medium. Control sections were processed in the same manner as experimental sections except that incubation with primary antibody was omitted.

METHODS Experimental tissues

Retindchoroid or cerebellar proteins were extracted in 2.3% SDS, 5% f3-mercaptoethanol, and 10%glycerol (v/v) in 0.062 M Tris-HC1, pH 6.8. The proteins were separated on 8% acrylamide gels with 4% stacking gels according to the method of Laemmli (1970) using the BioRad Mini Protean I1 gel system. Electrophoresis was carried out with 25 mA constant current until the protein samples entered the stacking gel and then the current was increased to 50 mA for the remainder of the run. Electrophoresis was done at room temperature. Prior to electroblotting, the gels were soaked in transfer buffer (20 mM Tris, 20% methanol (v/v), and 150 mM glycine) containing 1% SDS (w/v) for 15 minutes. Equilibration of the gels in this solution prior to electrotransfer improved the transfer of the proteins from the gel to the nitrocellulose membrane. The proteins were electroblotted onto nitrocellulose in transfer buffer without the added SDS for 2.5 hours at a constant voltage of 90 V, a t 4°C. Blots containing the transferred proteins were blocked in 10% normal goat serum (Vector laboratories) in Trisbuffered saline (TBS) (0.01 M Tris-HC1, pH 7.5) for 30 minutes prior to incubation with primary antiserum. Three antibodies were used in the immunoblot experiments, the GFAF' and vimentin monoclonal antibodies described above diluted 1:400 and 1:1,000, respectively, with TBS containing 10%normal goat serum, and an IFA monoclonal antibody used as an undiluted cell supernatant. The IFA antibody is a panspecific antibody that recognizes all intermediate filaments (Pruss et al., '81; Pruss, '85). Blots were incubated for 1hour in primary antibody. They were thoroughly washed in TBS (3 x 15 min) and then incubated for 1hour with alkaline phosphatase-conjugated antimouse IgG (Sigma) diluted 1500 with TBS containing 10% normal goat serum. Blots were again washed in TBS (3 x 15 min) and the binding sites visualized by developing in alkaline phosphatase substrate (100 mM NaC1, 5 mM MgCl,, 0.4 mM 5-bromo-4-chloro-3-indolyl phosphate, and 0.2 mM Nitro Blue tetrazolium in 100 mM Tris-HC1, pH 9.5) for 15 minutes in the dark. The reaction was terminated with distilled water and the blots were air-dried and photographed on Kodak Panatomic-X film. Control blots were processed in the same manner as experimental blots except that incubation with primary antibody was omitted.

Colonies of homozygous blind (rdlrd), heterozygous sighted (+/rd), and normal Rhode Island Red x Barred Rock sighted (+/+I breeder chickens are maintained at the University of Florida Animal Care Facility under NIH guidelines. The procedures used in these experiments were approved by the Institutionalhimal Care and Use Committee at the University of Florida. The eyes of 1, 7,21, and 33 days posthatch (dph) rd/rd, +/rd, and +/+ chicks were removed following decapitation and processed for either immunohistochemical or biochemical analysis following removal of the cornea and lens. A total of 24 chicks was used in this study, 2 chickdtime point/group. Eye cups designated for immunohistochemical study were fixed in 4% phosphate-buffered paraformaldehyde for 4 hours at 4°C. Following fixation, they were rinsed overnight in phosphatebuffered saline (PBS),pH 7.4. The retina and choroid of eye cups designated for biochemical analysis were scraped away from the underlying sclera after the pectin and vitreous had been removed. These tissues were then placed in plastic vials, frozen in liquid nitrogen, and stored at -70°C until use. In a few cases, chick cerebellar tissue was dissected from the brain and processed along with the eyes for immunohistochemical and biochemical analysis.

Immunohistochemistry Fixed tissues were embedded in 2% agarose in PBS and sectioned at 40 km on a Lancer Vibratome 1000. The Vectastain ABC system (Vector Laboratories) was used to localize the antigen in the tissue sections. Sections were blocked with PBS containing 1% bovine serum albumin (BSA) for 30 minutes and then incubated overnight at 4°C with mouse antiporcine monoclonal GFAP antibody (clone G-A-5, Sigma) diluted 1:lOOO with PBS containing 0.1% BSA and 0.25% Triton X-100. The GFAP monoclonal was originally characterized by Debus et al. ('83).Some sections were incubated overnight with a vimentin monoclonal antibody (clone D26, gift from Dr. Nancy Philp) diluted 1 5 0 with the PBS solution described above. All subsequent steps were carried out at room temperature. After 3 x 10

Immunoblots

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Fig. 1. Immunoblot of 33-day posthatch +/+ retindchoroid proteins treated with GFAF', intermediate filament, or vimentin monoclonal antibody. A. Total retinaichoroid protein stained with India ink according to the method of Hancock and Tsang ('83). B. Immunoblot stained with vimentin antibody. Arrowhead indicates the position of the 52 kD band. C . Immunoblot stained with intermediate filament antibody. Arrowheads indicate the positions of the 50 and 52 kD bands. D. Immunoblot stained with GFAP antibody. Arrowhead indicates the position of the 50 kD immunoreactive band. E. Control for antibody staining in which incubation with primary antibody was omitted.

RESULTS Control experiments A series of control experiments was conducted to examine the binding characteristics of the GFAP antibody under the conditions established for the immunoblot and immunohistochemical procedures. The specificity of the GFAP antibody for chick GFAP was assessed by comparing the GFAP antibody staining patterns with those obtained using the vimentin and anti-IFA antibodies. Immunoblots containing chick retindchoroid proteins probed with either GFAP, vimentin, or anti-IFA antibody showed that the GFAP antibody stained a single band (Fig. 1D) with an estimated M, of 50 kD, a value falling within the range of M, estimates reported for GFAP (Rueger et al., '79; Eng, '82). This result along with the observation that the same retinal protein band stained by the GFAP antibody was also stained by the IFA antibody (Fig. 1C) showed that the GFAP antibody was recognizing chick GFAP. Additional information concerning the binding specificity of the GFAP antibody was obtained by comparing GFAP and vimentin antibody staining patterns. The vimentin antibody stained two retinal protein bands (Fig. 1B) with estimated M, values of 52 and 43 kD, neither of which was stained by the GFAP antibody. Since the mass of chick vimentin has previously been estimated to be 52 kD (Brown et al., '761, and the 52 kD band was also stained by the IFA antibody (Fig. lC), the results indicated that the 52 kD protein recognized by the vimentin antibody was vimentin. The 43 kD protein band, which was stained by the vimentin and IFA antibodies, could represent a degradation product of vimentin. In addition to the immunoblot experiments, control studies were done to examine the effects of the immunohis-

tochemical fixation protocol on the ability of the GFAP antibody to recognize GFAP in situ. Cerebellum was chosen for these experiments because Bergmann glia in this tissue have been found to coexpress GFAP and vimentin in several animal species and are easily identified (Dahl et al., '81; Schnitzer et al., '81; Shaw et al., '81; Yen and Fields, '81; Debus et al., '83; Dahl et al., '85). Comparisons of the cerebellar immunohistochemical staining patterns obtained with the GFAP and vimentin antibodies showed that both antibodies decorated Bergmann glia processes (Fig. 2). GFAP-positive astrocytes were observed in both the granular layer and the white matter. Vimentin-positive astrocytes were observed in the white matter and stained astrocytic processes could be observed coursing through the granular layer. Cerebellar sections processed without primary antibody did not contain reaction product (data not shown). These results and the fact that a GFAP-positive 50 kDa band was detected on western blots of chick cerebellar proteins (Fig. 5 , lane 2) indicated that the fixation protocol used in the present study did not result in loss of the antigen and was not masking the antigenic sites recognized by the antibodies.

Retinal immunohistochemistry Tissue sections taken from central retina of 1, 7, 21, and 33 dph +I+, +/rd, and rdlrd chick were reacted with either vimentin or GFAP antibody. Control sections processed without primary antibody did not contain reaction product (Fig. 3A). Vimentin staining was present at all ages in +I+, +/rd, and rdlrd retina. A stained retinal section, representative of those observed, is shown in Figure 3B. Vimentin-positive, radially oriented filamentous profiles were present throughout each of the sections examined and extended from the nerve fiber Iayer to the outer limiting membrane. Heaviest staining was observed in the ganglion cell and nerve fiber layers. A similar pattern of staining was observed in peripheral retina (data not shown). The radial nature of the staining pattern and the fact that the stained cells possessed several of the attributes of chick Muller cells visualized with the Golgi method (Cajal, '73; Prada et al., '89) strongly suggested that Muller cells were the cells being stained by the antibody. In addition to the Muller cells, horizontal processes at the border between the inner nuclear layer and the outer plexiform layer were also decorated by the vimentin antibody. Vimentin staining of horizontal cells has been observed in rat, mouse, and cow (Drager, '83; Shaw and Weber, '83, '84). The fact that the chick staining pattern closely resembled that observed in rat, mouse, and cow retina suggests that chick retina may contain a horizontal cell type that expresses vimentin. Figure 4 shows representative retinal sections from 1, 7, 21, and 33 dph +I+, +lrd, and rdlrd chick, which were stained with GFAP antibody. GFAP staining was observed at all ages in +I+,+/rd, and r d r d retina and appeared to be restricted to the Muller cells. The Muller cells were stained throughout, but staining was most intense in those portions of the cell spanning the outer nuclear layer, the outer portion of the inner nuclear layer, and the ganglion cell and nerve fiber layers. Age-related increases in GFAP staining were observed in all three groups and were virtually indistinguishable. However, comparisons of the GFAP staining patterns observed in 21 and 33 dph retinas did reveal some differences in the distribution of GFAP staining within the Miiller cells in rdlrd retina that were not

GFAP EXPRESSION IN CHICK MULLER CELLS

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Fig. 2. GFAP and vimentin immunohistochemical staining of chick cerebellum. A. Section treated with the GFAP antibody. B. Section treated with the vimentin antibody. Arrows: a, astrocyte; b, Bergmann glia process. Magnification: 2.6 mm = 10 pm.

observed in +/+ and +/rd retina. In both 21 and 33 dph rdlrd retina, several of the Muller cell bodies located in the inner nuclear layer were clearly stained (Fig. 4K,L). In addition, increases in GFAP staining of the Miiller cell processes forming the outer limiting membrane and decreases in staining of that portion of the outer nuclear layer adjacent to the outer plexiform layer were observed in these retinas (compare Fig. 4G and K).

Retinal immunoblots The age-related increases observed in GFAP immunohistochemical staining of +/+, +/rd, and rdlrd chick retina were confirmed by immunoblot. For each of the three groups examined, increases in the intensity of GFAP staining of a single band with an estimated M, of 50 kD were observed with age (Fig. 5). No consistent differences in the intensity of staining of the GFAP immunoreactive band were observed between groups. It was noted that the staining intensities of the GFAP immunoreactive protein bands for 33 dph +/+, +/rd, and rdlrd retina/choroid, although greater than those observed for chick retinal choroid at the earlier time points, were considerably less than those observed for rat retina (Fig. 5, lane l), chick cerebellum (Fig. 5, lane 21, and rat cerebellum (Fig. 5, lane 31, despite the fact that the later samples contained only half as much protein as the chick retindchoroid samples. This finding was somewhat surprising in view of the magnitude of the increase in GFAP immunostaining observed in the 33 dph retinal tissue. Examination of stained SDS-PAGE gels following electrotransfer showed that there was always a small amount of Coomassie-stained protein in the 50 kD region of the gels containing the chick retina/ choroid proteins, which did not transfer to the membrane. Comparable regions of gels containing proteins extracted from rat retina, rat cerebellum, or chick cerebellum contained little to no stainable protein following transfer. Increasing the transfer time and/or altering the type and

pH of the transfer buffer did not seem to improve the transfer of the chick retinalchoroid 50 kD protein band.

DISCUSSION The present study was initiated to determine whether chick Muller cells accumulate GFAP in response to retinal degeneration induced by the rd mutation. The results of the immunohistochemical and immunoblot analyses of +I+, +/rd, and rd/rd retina lead to the following conclusions: 1. GFAP and vimentin are coexpressed in the chick Muller cell and are found throughout the cell and its processes. 2. Increases in GFAP expression are observed in +/+, +/rd, and rd/rd Muller cells with age. 3. Retinal degeneration in the rd chick retina does not significantly increase the expression of GFAP in the Muller cells. The present study is the first to demonstrate that GFAP is expressed in Muller cells of normal chick retina. To my knowledge, there has been only one previous report concerning GFAP in chick retina. In their study of the developmental distribution of vimentin in chick retina, Lemmon and Reiser ('83) mentioned that they were unable to detect GFAP in chick Muller cells. It is not clear why the results of these authors do not agree with those in the present study since information concerning the fixation protocol, immunohistochemical method, and GFAP antibody used in their GFAP experiments was not provided by these authors. Comparisons of the present results with the results of similar studies in mammalian and nonmammalian retina (Table 1)suggest that, with respect to GFAP expression in normal retina, chick Muller cells may be more like goldfish and frog Muller cells than mammalian Muller cells. Very few studies have been done to examine GFAP expression and distribution in nonmammalian retinas. As in the chick retina, analyses of normal goldfish and frog retina (Table 1)

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retina and rat and chick cerebellum. This observation, along with Dahl and Bignami's ('73) immunodiffusion data, suggest that the GFAP protein in chick retina may possess biochemical properties that distinguish it from the GFAP protein expressed in chick brain, rat retina, and rat brain. Several of the mammalian studies listed in Table 1 reported the presence of detectable amounts of GFAP within Muller cells. This was somewhat surprising and seemed to contradict the view that Muller cells in normal mammalian retina do not express GFAP. For example, only one of six studies of cat retina failed to detect GFAP immunoreactivity in cat Muller cells (Shaw and Weber, '84). Karschin et al. ('86)observed slight GFAP immunoreactivity in the basal processes of the Muller cells in a few of their wholemount preparations, but their data were not strong enough to support a definite conclusion. Taken together, these results suggest that the generalization that RPE- 1 OLMMuller cells do not contain GFAP in normal mammalian ON Lretina may be too broad. OPL- ' Lack of unanimity among the published studies of retinas of specific mammalian species may be due to a combination of factors including immunocytochemical detection method, INLfixation protocol, binding characteristics of individual antibodies and region-specific differences within retina. Several research groups have commented on the effects of various fixation protocols on immunohistochemical detection of GFAP in retina (Ontenienteet al., '83; Bjorklund and Dahl, IPL'85; Nona et al., '89; Vaughan et al., '90). For example, Bjorklund and Dahl ('85) found that GFAP staining of Muller cells in rat retina was best if the retinas were fixed with paraformaldehyde and virtually absent in acetone GCLNFL..-..,,.,.. . -. . , , .... fixed retinas. Their observations contrast with those reFig. 3. Vimentin immunohistochemicalstaining of 1day post-hatch cently made by Vaughan et al. ('90) who found that fixation +/+ chick retina. A. Control section in which primary antibody was with paraformaldehyde drastically reduced detection of omitted during the immunohistochemicalprocedure. B. Retinal section GFAP epitopes in rabbit retina and advocate the use of treated with vimentin antibody. Arrows: a-horizontal processes deco- unfixed retina in GFAP studies. The conclusions reached by rated with the vimentin antibody; L v i m e n t i n immunoreactive radial Bjorklund and Dahl ('85) and Vaughan et al. ('90) do not processes in Muller cells; c-immunoreactive Muller cell endfeet. Abbreviations: RPE, retinal pigment epithelium; OLM, outer limiting readily explain the inconsistencies found in the literature (see Table 1).The essential feature of effective formaldemembrane; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell hyde fixation is the formation of chemical cross-links layer; NFL, nerve fiber layer. Magnification: 2.6 mm = 10 Fm. between the end-groups of adjacent protein chains. With washing, many of the interactions between formaldehyde and protein are reversed, leaving the majority of the protein suggest that the Muller cells in these species express GFAP active groups in a condition to react with other reagents in adult life. In addition, the pattern of GFAP immunostain- (Pearse, '80). In contrast, treatment of proteins under ing within the Muller cells in goldfish retina closely resem- extreme pH conditions or high concentrations of organic bles that seen in chick retina. In both species, GFAP solvents (e.g., acetone) has been shown to disrupt their immunoreactivity is distributed throughout the processes hydrophobic bonds and results in variable loss of high-order of the Muller cells (Bignami, '84; Nona et al., '89), a protein structure, which may or may not be reversible situation unlike that found in most mammalian species (Pearse, '80). Clearly, the different actions of specific where GFAP immunostaining, if present, is limited to the fixatives on proteins could influence their immunohisregion of the cell forming the inner limiting membrane. tochemical detection; however other factors must also Although polyclonal antibodies directed against mamma- contribute to the differences observed between studies. Another factor that is likely to influence the outcome of lian GFAP have been successfully used in the goldfish and frog studies, it has been suggested that the protein or immunohistochemical studies is the method used to visualproteins in the Muller cells of these animals that react with ize the antigeniantibody complex within the tissue. Recent these antibodies may not be identical to mammalian GFAP advances in immunocytochemical methods, which include (Jones and Schechter, '87; Szaro and Gainer, '88). Along the development of the avidin-biotin-peroxidase (ABC) this same line, Dahl and Bignami ('73) noted that chick technique, have greatly improved detection of antibody1 GFAP, although possessing the same extraction properties antigen complexes. Indirect imniunofluorescent techniques and molecular weight as human GFAP, produced an do not possess the signal amplification characteristics of the Ouchterlony immunodiffusion pattern suggesting incom- peroxidase anti-peroxidase (PAP) and ABC techniques. plete identity with human GFAP. In the present study, it Thus it is possible that GFAP in Muller cells of normal was noted that the electrotransfer characteristics of the mammalian retina could be overlooked if the amount of GFAP-containing 50 kD protein band differed from compa- GFAP in these cells is low and indirect immunofluorescent rable regions of gels containing proteins extracted from rat techniques are employed. The fact that several investiga-

L

~

I

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Fig. 4. GFAP immunohistochemical staining of +/+ (A-D), +/rd (E-H), and r d r d (I-L) retina at 1(A, E,I), 7 (B,F, J), 2 1 (C, G , K), and 33 (D, H, L) days posthatch. Increases in GFAP staining were observed in all three groups with age. Differences noted in 21 and 33 rdird retina ( K and L) included staining of several Muller cell bodies (arrows),

increased staining of the OLM and decreased stainingin the inner half of the ONL. Abbreviations: OLM, outer limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Magnification: 2.6 mm = 10 km.

tors have detected the relatively large amounts of GFAP present in retinal astrocytes and in Muller cells of degenerating retina using indirect immunofluorescent techniques supports this idea (Bignami and Dahl, '79; Drager and Edwards, '83; Eisenfeld et al., '84; Shaw and Weber, '84; Eisenfeld et al., '87; Sarthy and Fu, '89). Perhaps one of the

reasons Shaw and Weber ('84) were unable to detect GFAP in cat Muller cells is the fact that they used indirect immunofluorescent techniques in their study (Table 1). Detection of GFAP in Muller cells may also be affected by the antigenic determinants recognized by individual antibodies (Table 1) and region-specific differences in retina. The

588

S.L. SEMPLE-ROWLAND 1 DPH

a

b

c

a

7 DPH b c

21 DPH

a

b

33 DPH c

a

b

c

1

2

3

-110 -84

-47

Fig. 5. Immunoblot showing increase i n GFAP staining with increasing age i n +i+ (a), +ird (b),a n d rdird (c) retinakhoroid. Staining of a single band at 50 kD was first evident i n the retinal samples at 7 days posthatch (dph). Lanes a, b, a n d c were loaded with 80 kg total retinal protein. Lane 1-albino rat retina protein. Lane 2-33 dph +i+ chick cerebellar proteins. Lane 3-albino rat cerebellar proteins. T h e GFAP antibody stained a single band of approximately 50 kD i n lanes 1-3. Lanes 1-3 were loaded with 40 pg total protein.

TABLE 1. Muller Cell GFAF' Expression in Normal Retinas of Various Animal Species Miiller S t a i n Species

Yes

Human

No

Fix'

Antibody, a n t i g e n , o r cloneisourceimethod'

Reference

X

carn 10%f 10%f pf+g3 4%pf a 4%pf pf+g3 pf+g3 4%pf

pc/theirsPAP pciDr. L. EngiPAP pciDakolPAP pciDakoiSEAG mcilabsystems; pciDr. RaffiABC-F pc,pig spinal cord (Sharp et al., '82)itheirsilI pciDrs. D. Dahl and A. BignamiPAP pciantihovine GFAPiDakolIG pc,antihovine GFAPiDakoiSEG pciDakoiPAP pc,pig spinal cord (Sharp et al., '82)itheirsiIl pcDrs. D. Dahl and A. BignamiiII mc,clone G-A-5 (Debus et al., '83)iBoehringeriII mciLahsystemd pcDr. Raff/ABC-F pc,human/not giveniII pciDako; mclAmershamiII mc,human,clone 6FZiSynbioiABC pc,humaniDr. A. Bignami; pc (Sharp et al., '82)iII pc (Dahl and Bignami, '76)itheirslII pc (Eng and Ruhinstein '78YtheirsiPAP pc,humaniDr. L. E n d 1 mc,clone G-A-5 (Debus et al., '83YBoehringeriABC pqantihovine GFAPDr. L. EngiABC mcilabsystems; pciDr. RaffiABC-F pc (Dahl and Bignami, "76)itheirslII pc,pig spinal cord (Sharp et al., '82)itheirslII pc (Bignami and Dahl, '74)iDr. A. BignamUII pclDakoPAP pc (Fedoroff et al., '83)itheirsiII pciDr. L. EngiII mc,clone G-A-5 (Debus et al., '83)lSigmdABC pc,antihuman/Dr. R. PrussiPAP pc,human (Dahl and Bignami, '76)itheirsiII pciAccurate Biochemicals or Dr. D. DahUII pc,antihumanDr. Dahl; mc,antiporcine/ICNiPAP uc.antihuman and antibovine GFAPiDakoiABC

Kumpulainen et al., '83 Molnar et al., '84 Hiscott et al., '84 Guerin et al., '90 Stone and Dreyer, '87 Shaw and Weber, '84 Karschin et al., '86 Erickson et al., '87 Lewis et al.,. '89 Ekstrom e t al., '88 Shaw and Weber, '84 Schnitzer, '85 Schnitzer, '87 Stone and Dreher, '87 Eisenfeld et al., '87 Vaughan et al., '90 Durlu et al., '90 Shaw and Weher '83, '84 Bignami and Dahl, '79 Dixon and Eng, '81 Eisenfeld et al., '84 Seiler and Turner, '88 Penn et al., '88 Stone and Dreher, '87 Bjorklund and Dahl, '85 Shaw and Weher, '84 Bromberg and Schachner, '78 Ekstrom et al., '88 Abd-El-Basset et al., '88 Sarthy and Fu, '89 present study Szaro and Gainer, '88 Bignami, '84 Linser et al., '85 Nona et al., '89 Jones and Schechter, '87

X X

Monkey Cat

X X X

? X X X

Rabbit

X

a

X

X

4%pf 4%pf 4%pf 4%pf none Pf a a Pf 4%f 4%pf 4%pf 4%pf a,pf5 a a 4%pf 4%pf 4%pf 4Bpf 4%pf

X

a

X

4%pf 47cpf 4%Df

X X

X

X X X

Rat X

X X

Mouse

X X

X X X

Chicken Frog Goldfish

X

X

X

.

I

'Fixation methods: a = acetone, pf = paraformaldehyde, f = formaldehyde, g = glutaraldehyde, carn = Carnoy's (chloroformimethanoUaceticacid) 'pc = polyconal, mc = monoclonal, antigen source and reference concerning production and characterization of antibody is listed if given/ immunohistochemical procedure: I1 = two step indirect immunofluorescent system (e.g., FITC), PAP = three-ste peroxidase-antiperoxidasesystem, ABC = three-step avidin-biotin system, ABC-F = three-step avidin-biotin fluorescent system, SEG = silver-enhanced go& label, IG = immunogold. 'Fixative was 1% pf + 1% g. 'Authors tested several fixatives: 4%, f, 2% pf, 2%pf + 50% ethanol, 95% ethanol, and Perfix (Fisher Scientific) 'Authors used either acetone or parakrmaldehyde fixative.

GFAP EXPRESSION IN CHICK MULLER CELLS source of the antigen used to generate antibodies, as well as whether monoclonal or polyclonal antibodies are used, can influence binding characteristics. Evidence that GFAP immunostaining results can, in part, be determined by the antibody employed was obtained by Bjorklund and Dahl ('85). Using a polyclonal antiserum to GFAP raised in rabbits against degraded human antigen (Dahl and Bignami, '76) and monoclonal GFAP antibodies raised against cytoskeletal preparations of chick brain, these investigators found that the Muller cells in paraformaldehyde fixed normal rat retina stained positively for GFAP using the polyclonal antiserum but did not stain using the monoclonal antibodies. Finally, several investigators have noted that there is some variability in the expression of GFAP in Muller cells located in different regions of the retina. In both human (Hiscott et al., '84) and mouse retina (Bromberg and Schachner, '78; Drager and Edwards, '83; Sarthy and Fu, '891, Muller cells located near the ora serrata have been observed to stain positively for GFAP while little or no staining was found in Muller cells located in central retina. In the present study, Bergmann glia and astrocytes in chick cerebellum were found to stain positively for both vimentin and GFAP. This result by itself was not surprising in view of the fact that mammalian Bergmann processes seem to invariably coexpress vimentin and GFAP (Dahl et al., '81; Schnitzer et al.,'81; Shaw et al., '81; Yen and Fields, '81). However, the results of previous studies of chick cerebellum have suggested that chick Bergmann glia contain vimentin but not GFAP (Debus et al., '83; Dahl et al., '85). Comparisons of these studies with the present one revealed that both Debus et al. ('83) and Dahl et al. ('85) used indirect immunofluorescence to detect GFAP in cerebellar tissue that was briefly fixed with acetone. Based on the previous discussion and the fact that the GFAP antibody employed by Debus et al. ('83) was used in the present study, it seems likely that the discrepancies observed between these earlier studies and the present one are due to differences in fixatives and/or immunocytochemical detection methods. Arecent report by Shehab et al. ('90) showing that GFAP staining of Bergmann glia is enhanced in rat cerebellar tissue fixed with 4% paraformaldehyde and virtually absent from tissue fixed in acid-alcohol (5% acetic acid, 95% ethanol) supports this conclusion. A major objective of the present study was to determine if Miiller cells accumulate GFAP in chick retinas affected by the rd mutation. The fact that increases in Muller cell GFAP were observed in normal as well as degenerating chick retinas suggests that GFAP may be important for the normal functioning of chick Muller cells. Comparisons of the GFAP staining patterns of +/+, +/rd, and rd/rd retina in the present study revealed a change in the distribution of GFAF' within the Muller cells of rd/rd retina following the onset of degeneration. In 21 and 33 dph rd/rd retina, increased GFAP staining was observed in the region of the Muller cell bodies and in the processes of the Muller cells forming the outer limiting membrane. The increased staining observed in the cell bodies of the Muller glia in these retinas could result from redistribution of the GFAP protein within the Muller cell and/or an increase in the synthesis of GFAF' within the cell body region. Increased GFAP immunostaining in degenerating mammalian retina has been associated with increased transcription of the GFAP gene (Sarthy and Fu, '89). Thus the increased staining of the Muller cell bodies in degenerating rd chick retina could result from increased activation of the GFAP

589

gene in response to degeneration. If this turns out to be the case, then the signals responsible for initiating the dramatic increases in GFAP in the Muller cells of degenerating mammalian retina may also be present in chick retina. Further studies of the biochemical and molecular mechanisms underlying GFAP expression in normal and rd chick retina should improve our understanding of the regulation of these intermediate filaments in normal as well as degenerating tissue.

ACKNOWLEDGMENTS I thank Dr. Wolfgang J. Streit and Laura Errante for their helpful discussions during the course of this study and their critical reading of the manuscript. In addition, I thank Hanke van der We1 for her excellent technical assistance, Dr. Robert J. Ulshafer for financial assistance in the care and maintenance of the animals, and Dr. Nancy Phelp for providing the vimentin monoclonal antibody. This work was supported in part by NIH Grants R 0 1 EY08340 (SLS-R),R01 EY04590 (RJU), and a grant-in-aid from The Fight For Sight Research Division of the National Society to Prevent Blindness (SLS-R).

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