Exp. Eye Res. (2001) 72, 495±501doi:10.1006/exer.2000.0984, available online at http://www.idealibrary.com on

Cellular and Subcellular Patterns of Expression of bFGF and CNTF inthe Normal and Light Stressed Adult Rat Retina

NATALIE WALSH, KRISZTINA VALTER AND JONATHAN STONE*

NSW Retinal Dystrophy Research Centre, Department of Anatomy and Histology, University of Sydney,Sydney, NSW 2006, Australia

(Received Cleveland 24 August 2000, accepted in revised form 5 December 2000 and published

subcellularetina. Re

0014-483

* Addressand HistoloE-mail: jons

electronically 16 March 2001)

This study compared the distributions in the normal and light stressed rat retina of the neuroprotectivefactors bFGF (basic ®broblast growth factor) and CNTF (ciliary neurotrophic factor). Albino Sprague±Dawley rats were raised in cyclic light and some were exposed to bright continuous light for 48 hr, toinduce light damage of photoreceptors. Their retinas were prepared as cryosections, immunolabelledwith antibodies to bFGF and CNTF and analysed by confocal microscopy. Both factors were prominent inmacroglial cells (astrocytes, MuÈ ller cells) and the retinal pigment epithelium (RPE). In the somas of thesecells the distributions of the two factors were complementary, with bFGF concentrated in the nuclei andCNTF in the cytoplasm. Both factors were distributed along the processes of macroglial cells, in granularform. CNTF was not detected in neurones, but bFGF was consistently present in the cytoplasm ofganglion cell somas and, in regions of retina subject to stress, in the cytoplasm of photoreceptors. bFGFwas not detected in the nuclei or processes of neurones. In retina stressed by light exposure or proximityto the anterior edge of the retina, the levels of bFGF and CNTF were up-regulated, without majorchanges in localization. Macroglial cells (MuÈ ller cells, astrocytes) play a major role in distributing bFGFand CNTF throughout the retina. The different localizations of the two factors within the somas ofmacroglial, RPE and photoreceptor cells, suggest that their protective actions are exerted by distinctive

#

mechanisms. 2001 Academic PressKey words: basic ®broblast growth factor; ciliary neurotrophic factor; light damage; macroglia; retinal

pigment epithelium.

much more prominent in neurones, particularly

1. Introduction

The degeneration of photoreceptors, the key event inmost retinal degenerations, is prevented or slowed bya number of trophic growth factors, particularly basic®broblast growth factor (bFGF or FGF2) and ciliaryneurotrophic factor (CNTF) (LaVail et al., 1992;Cayouette et al., 1998). Both factors protect photo-receptors if applied to the retina exogenously, byintravitreal injection (Faktorovich et al., 1990, 1992;Lin et al., 1997) or viral mediated delivery (Akimotoet al., 1999; Lau et al., 2000) and both are expressedby the retina endogenously (Gao and Holly®eld, 1996;Kirsch et al., 1997; Ju et al., 1999; Chun et al.,2000). Further, the endogenous expression of bothis up-regulated by stress on the retina, whethercaused by light exposure (Steinberg, 1994; Gao andHolly®eld, 1996; Liu et al., 1998) or by mechanical(Cao et al., 1997b) or laser-induced (Chu et al., 1998;Xiao et al., 1998) trauma. We have used immuno-labelling to detect and compare the retinal distri-butions of bFGF and CNTF at the cellular and

r levels, in normal and light-stressed ratsults show that both bFGF and CNTF are

5/01/050495�07 $35.00/0

correspondence to J. Stone, Department of Anatomygy, University of Sydney F13, NSW 2006, Australia.tone@anatomy.usyd.edu.au

found within the macroglial cells of the retina(astrocytes and MuÈ ller cells) and the retinal pigmentepithelium (RPE), and that bFGF is also found inretinal neurones. Most interesting were two differ-ences observed in the cellular distributions of the twofactors. First, the distributions of the two factorswithin astrocytes, MuÈ ller cells and RPE cells, werecomplementary: bFGF was found principally withincell nuclei, CNTF within cytoplasm. Second, bFGF was

photoreceptors.

2. Materials and Methods

Data from three separate experiments were used.All animals were albino Sprague±Dawley rats bornand raised in dim (5±10 lux) cyclic light. In eachexperiment, two animals (P70) were exposed to brightcontinuous light (BCL, 1400±1800 lux) for 48 hrand littermates served as controls. The control animaland one of the light-exposed animals were killed at theend of the 48 hr period and the third animal waskilled 1 week later with an overdose of sodiumpentobarbitone (60 mg kgÿ1 intraperitoneal). Thesuperior aspect of the eye was marked and the eyes

were then removed and immersion-®xed in 4 %paraformaldehyde for 1±3 hr. After three rinses in

# 2001 Academic Press

0.1 M PBS the eyes were left overnight in a 15 %sucrose solution to provide cryoprotection. Eyes were

496

embedded in mounting medium by snap-freezing inliquid nitrogen and were cryosectioned at 20 mm.

The sections were then washed and coverslipped in agelatin±glycerol mixture.

Immunohistochemistry

Sections were labelled with a mouse monoclonalantibody against a biologically active region of bovinebasic FGF (Type I, Upstate Biotechnology, Lake Placid,NY, U.S.A.) and with either a rabbit polyclonal anti-body to CNTF (Chemicon International, Temecula,CA, U.S.A.) or a rabbit polyclonal antibody to GFAP(BioGenex, San Ramon, CA, U.S.A.). All primary anti-bodies are highly speci®c for rat tissue. The secondaryantibody for bFGF was an Alexa 488-conjugated goatanti-mouse IgG (H � L), the CNTF secondary antibodywas Alexa 594-conjugated goat anti-rabbit IgG (H � L)(Molecular Probes, Portland, OR, U.S.A.) and thesecondary antibody for GFAP was a Cy3-conjugatedanti-rabbit IgG (Sigma, St. Louis, MO, U.S.A.).

Unless otherwise stated all washes were for2 � 5 min in 0.1 M PBS and were performed atroom temperature while incubations were at 378C.Sections were brought to room temperature andwashed. To allow penetration and blocking of the

tissue, slides were placed in a 0.3 % Triton : 1 % BSAsolution for 15 min. The bFGF and CNTF primary

FIG. 1. The distribution of bFGF and CNTF in macroglia (Muimmunolabelling for bFGF is shown in green. In (A)±(H) and (J)±label shows immunolabelling for GFAP. (A) In the inner nuclear labe MuÈ ller cells. The bFGF� pro®les are always `®lled' suggestinggranules are found in processes extending from the bFGF� nuclei(arrows) suggesting that CNTF is present in MuÈ ller cell cytoplaphotoreceptors are bFGFÿ. This shot was chosen to show the occaThe bFGF is in the cytoplasm, leaving the nucleus bFGFÿ. Againextend through the outer layer. (C) and (D) In the outer nuclearprocesses, extending to where these processes coalesce to form thebFGF granules are also present in these radial processes and rtangentially oriented processes. (E) At lower power CNTF and bFGlayer (opl), and in radial and tangential processes of MuÈ ller cells.cytoplasm of ganglion cells, and absent from their nuclei. CNTF isinner plexiform layer (ipl) and in astrocytes at the inner surface. b(G) Three astrocyte nuclei (green/yellow, one is arrowed) are sprocesses are CNTF� and entwine to form the ring-shaped glia limcontributing to the inner limiting membrane. Within the glia limCNTFÿ. The nuclei of astrocytes are yellower than of MuÈ ller cells inucleus. The inset in (G) shows one astrocyte (nucleus arrowed) wthe nucleus site. (H) At the inner retinal surface, bFGF is prominenuclei or processes. The green-yellow nucleus of an astrocyte issurface and between ganglion cell somas. (I) Near the edge of thereaching a maximum at the edge (arrow). In parallel, GFAP immuprominent in the radial processes of MuÈ ller cells. (J) and (K) Centra(K), bFGF is much more prominent than in central retina (J) moprominent at the edge, especially in the radially oriented processebFGF is prominent in the nuclei of MuÈ ller cells and CNTF in their cedge, bFGF concentrates in the cytoplasm of photoreceptors, not inthe retina (green) and from central retina (black) measured alongis about half as thick as central retina; the trace lengths have beensignals from the edge of the retina (red) and from central retina (blshow the following: (B) 5 mm for (A) and (B); (C) 5 mm for (C) and100 mm; (J) 25 mm for (J) and (K); (L) 10 mm and (M) 5 mm.

antibodies were made up in the Triton : BSA solutionat a dilution of 1 : 200, while the GFAP was in a 1 : 1dilution of the same solution. Sections were incubatedwith a mixture of two antibodies (i.e. either bFGF andCNTF or bFGF and GFAP) for 1 hr at 378C. Sectionswere washed, then incubated for 1 hr in thesecondary antibodies diluted 1 : 200 in 0.1 M PBS.

N. WALSH ET AL.

Measurement of Labelling

For semiquantitative comparison of immuno-labelling, sections to be compared were ®xed by thesame protocols, cut in the same session and labelled inthe same runs, with identical incubation and proces-sing times. Photomultiplier settings on the confocalmicroscope were optimized to cover the range oflabelling to be studied and then held constant.Excitation and barrier ®lters were adjusted toeliminate cross talk between the red and greenemissions. Confocal images were studied with theAnalysis tool of NIH Image. Transects 9-pixel wideacross the thickness of the retina were delineated andthe labelling pro®le, averaged over the width of the

line, were captured separately for red and greensignals.

È ller cells, astrocytes) of the rat retina. In all of (A)±(M),(M), CNTF immunolabelling is shown in red. In (I) the red

yer (i) bFGF is prominent in a subclass of somas, presumed tothat bFGF ®lls the nucleus. Red (CNTF) and green (bFGF)and CNTF granules are also scattered around each nucleussm. (B) In the outer nuclear layer in central retina most

sional bFGF� soma present in this layer in unstressed retina.bFGF and CNTF granules are found along processes whichlayer, CNTF granules (red) concentrate in radially orientedouter limiting membrane [between the arrows in (D)]. Greened and green granules are found [arrows in (C)] in moreF (red and green) granules are present in the outer plexiform(F) In the inner half of the retina, bFGF is prominent in themost prominent in radial MuÈ ller cell processes crossing the

FGF� pro®les are again prominent in the inner nuclear layer.paced around a vessel at the inner retinal surface. Theiritans, and also spread along the inner surface of the retina,itans, the vessel's endothelial cells are bFGF� (green) and

n these preparations, suggesting the presence of CNTF in theith the bFGF signal removed, to show a CNTF-poor region atnt in the cytoplasm of ganglion cell somas, but not in theirseen at top. Its CNTF� processes spread along the retinal

retina, the level of bFGF in the outer nuclear layer (s) rises,nolabelling increases (red signal) and near the edge GFAP isl and peripheral regions of the normal rat retina. At the edgest markedly in the outer nuclear layer. CNTF is also mores of MuÈ ller cells. (L) In the inner nuclear layer at the edge,

ytoplasm and processes. (M) In the outer nuclear layer at thetheir nuclei. (N) bFGF immunolabel signals from the edge of

a transect across the thickness of the retina. Peripheral retinanormalized, to facilitate comparison. (O) CNTF immunolabel

ack), measured along the same transects as in (N). The scales(D); (E) 10 mm for (E) and (F); (G) 25 mm for (G) and (H); (I)

FIG. 1. A±O. For legend see facing page.

LOCALIZATION OF bFGF AND CNTF IN RAT RETINA 497

bFGF remained prominent in the nuclei of macroglial

3. Results

Cellular Localizations of bFGF and CNTF in NormalRetina

In MuÈ ller cells bFGF immunolabelling was promi-nent in the cells' nuclei in the inner nuclear layer[green in Fig. 1(A)] and was detectable, as granules,in processes in the inner [Fig. 1(A)] and outer[Fig. 1(B)±(D)] nuclear layers. CNTF immunolabellingwas prominent, as granules, in MuÈ ller cell processesand surrounding MuÈ ller nuclei in the inner nuclearlayer [red in Fig. 1(A)] and in MuÈ ller processes in theouter nuclear layer [Fig. 1(B) and (C)] and formingthe outer limiting membrane [Fig. 1(D)]. CNTF wasnot detected in MuÈ ller cell nuclei. At low magni®ca-tion, CNTF appeared to concentrate in radiallyoriented processes [Fig. 1(E)]. bFGF was found inthese radial processes but also in the processes whichsurround each soma of the outer nuclear layer. Athigher magni®cation it is apparent that CNTF� (red)and bFGF� (green) granules are intermingled alongall MuÈ ller processes in the ONL [arrows in Fig. 1(C)],both tangential and radial. In the inner plexiform layer(F) CNTF concentrated in the processes of MuÈ ller cells.

In astrocytes, as in MuÈ ller cells, CNTF was found inthe cytoplasm of the soma and in the cells' processes[Fig. 1(G) and (H)]. The CNTF-immunolabelling ofastrocyte cytoplasm and processes appeared continu-ous rather than granular, suggesting that CNTF levelsare higher in astrocytes. Morphologically, CNTFlabelling demonstrated the processes of astrocytesforming the glia limitans of a vessel [Fig. 1(G)] andstretching along the inner surface of the retina[Fig. 1(F)±(H)]. bFGF was prominent in astrocytenuclei, making the nuclei readily identi®able as brightgreen pro®les [Fig. 1(F)±(H)]. However, the nuclei ofastrocytes appear yellower than MuÈ ller cell nuclei [Cf.Fig. 1(A) with (G) and (H)], indicating a signi®cantlevel of CNTF in or around astrocyte nuclei. Never-theless, the level of CNTF in astrocyte nuclei was lowerthan in their cytoplasm. This is apparent in the inset inFig. 1(G), which shows the soma of one astrocyte(arrowed) with the bFGF labelling deleted. Inside theglia limitans of the vessels, bFGF was prominent in theendothelial cells forming the wall of the vessel[Fig. 1(G)]. CNTF was not detected in endothelial cells.

In retinal neurones, CNTF labelling was incon-spicuous. bFGF was clearly present in some neurones,most clearly ganglion cells [Fig. 1(F) and (H)] andphotoreceptors [Fig. 1(B), (I), (K) and (M)]. bFGFlabelling was reliably detectable in ganglion cellsomas but, in photoreceptors, bFGF labelling variedmarkedly with light history and location (Stone et al.,1999). In central regions of retinas exposed to onlylow light levels, photoreceptors were largely free ofbFGF, though occasional somas were clearly labelled,as in Fig. 1(B). Near the anterior edge of the retina

498

(independent of light experience) the level of bFGF inphotoreceptor somas rose sharply, to a maximum at

the extreme edge [Fig. 1(I), (K) and (M)]. Where bFGFwas present in neurones it was found in the cytoplasmof somas, and not in nuclei or neuronal processes(dendrites, axons).

The increase in bFGF levels in photoreceptors somasat the anterior edge of the retina [Fig. 1(I) and (K)]has been described previously (Xiao et al., 1998;Stone et al., 1999). It is paralleled by an increase inthe expression of the intermediate ®lament proteinGFAP by retinal macroglia [Fig. 1(I)]. The appearanceof GFAP in the radial processes of MuÈ ller cells,apparent in Fig. 1(I) towards the edge of the retina(arrow), is an established index of environmental(Eisenfeld, Bunt-Milam and Vijay Sarthy, 1984; Burnsand Robles, 1990; Raad et al., 1996), genetic(Eisenfeld et al., 1984; Ekstrom et al., 1988) andmechanical (BjoÈrklund, Bignami and Dahl, 1985;Erickson et al., 1987; Humphrey et al., 1993; Caoet al., 1997c) stress to the retina. Immunolabelling forCNTF also increased towards the edge of the retina[Cf. Fig. 1(J) with (K)]. CNTF appears to ®ll theprocesses of MuÈ ller cells near the edge of the retinaand bFGF ®lls the cytoplasm of every photoreceptorsoma [Cf. Fig. 1(K) and (M) with (B)]. CNTF levels inthe outer limiting membrane also appeared raisedtowards the edge of the retina [Cf. Fig. 1(M) and (D)].

The up-regulation of CNTF and bFGF proteinexpression at the anterior edge of the retina is shownquantitatively in Fig. 1(N), for bFGF and (O) for CNTF.CNTF is up-regulated at the ILM and through the IPLand again in the OPL. CNTF is not up-regulated in theONL, but is sharply up-regulated in the outer limitingmembrane. By contrast, bFGF is up-regulated in theinner plexiform and inner nuclear layers, andthroughout the outer nuclear layer, but not in theouter limiting membrane. The up-regulation of CNTFand bFGF at the anterior edge of the retina did notgreatly affect their cellular localization. In particular,

N. WALSH ET AL.

cells and CNTF in their cytoplasm.

Regulation by Light Damage

Regions of mid-peripheral retina from a controlanimal, from an animal exposed to 48 hr BCL andfrom an animal which survived exposure to 48 hr BCLby 1 week are shown in Fig. 2(A)±(C). Light damageinduced increases in the levels of both bFGF and CNTFabove control levels. Increases in bFGF levels weremost prominent in the cytoplasm of ganglion cellsomas and in the cytoplasm of photoreceptor somas[Fig. 2(A)±(C)]. Increases in CNTF levels were mostprominent in astrocytes at the inner surface of theretina, in the radial process of MuÈ ller cells and at theouter limiting membrane. When the immunolabellingsignals for CNTF and bFGF were separated [Fig. 2(D)]the tendency for CNTF to remain concentrated in the

radial processes of MuÈ ller cells, and for bFGF to enterthe cytoplasm of photoreceptor somas, was clear.

FIG. 2. (A)±(F) Effects of light damage on bFGF and CNTF levels. (A)±(C) The levels of bFGF and CNTF in central retinaincreased when the retina was exposed to bright continuous light (BCL) for 48 hr. Photomultiplier settings were kept constantto make the images comparable. (D) The CNTF (red) and bFGF (green) labelling of the same patch of central retina, withphotomultiplier settings set to demonstrate low level labelling. (E) CNTF immunolabelling signals from the retina whichsurvived exposure to BCL by 1 week (red) and from a control retina (black). (F) bFGF immunolabelling signals from the retinawhich survived exposure to BCL by 1 week (green) and from a control retina (black). (G)±(J) RPE cells. (G) and (H) The nuclei ofRPE cells are strongly bFGF�, while their cytoplasm is strongly CNTF�. Bruch's membrane and the vessels of thechoriocapillaris label strongly for bFGF. (I)±(J) At the edge of normal retina and in a retina exposed to BCL for 48 hr, CNTFlabelling of the cytoplasm of RPE cells appears strong, but bFGF labelling of their nuclei was, unexpectedly, absent or weak. The

(G);

LOCALIZATION OF bFGF AND CNTF IN RAT RETINA 499

The up-regulation of bFGF and CNTF induced bylight damage is shown quantitatively in Fig. 2(E) and(F). The up-regulation of CNTF was prominent at theILM, in the IPL and INL and at the OLM but limited inthe ONL. Conversely, the up-regulation of bFGF was

scales represent the following: in (C) 50 mm for (A)±(C) and

most prominent in the ONL and in ganglion cellsomas [Fig. 2(F)].

CNTF and bFGF in the RPE

In the RPE, as in the somas of astrocytes and MuÈ llercells, bFGF was prominent in nuclei and CNTF in

cytoplasm [Fig. 2(G) and (H)]. At the edge of the retina

in (D) 20 mm; in (H) 10 mm and in (I) 10 mm for (I) and (J).

[Fig. 2(I)] and in light damaged retina [Fig. 2(J)] CNTFlevels were high, with some evidence of up-regulation,

compared to more central regions of retina. Surpris-ingly, both at the edge of the retina [Fig. 2(I)] and in

500

light-damaged retina [Fig. 2(J)], the level of bFGF in the

mRNA at the subcellular level, in an attempt to de®ne

Foundation of the University of Sydney and the National

nuclei of RPE cells seemed lower than normal retina.

4. Discussion

The cellular distributions of bFGF and CNTF showcommon and distinct features, which may providesome insight into their functional roles. In neuralretina unstressed by light experience or other damage,both factors are found predominantly in the macrogliaof the retina, astrocytes and MuÈ ller cells. Both aredistributed in granular form within the processes ofMuÈ ller cells, through all layers between the ILM andOLM. Both are strongly expressed by RPE cells. Theexpression of both in macroglia is up-regulated bystress, con®rming a number of previous reports (Stone,Makarov and Hollander, 1995; Cao et al., 1997a,b;Liu et al., 1998). The wide retinal distribution of thesefactors and their up-regulation by stress ®t well withtheir known protective actions on retinal neurones(Faktorovich et al., 1992; LaVail et al., 1992).

The present results con®rm Ju et al.'s (1999)description of CNTF in the processes of MuÈ ller cellsand astrocytes, and their observation that CNTF levelsare up-regulated by stress, in their case ischaemicstress (Ju et al., 1999). Kirsch et al. (1997) alsoreported that CNTF is most prominent in MuÈ ller cells.On the other hand, the present results and those of Juet al. (1999) differ from Bajetto, Schettini and Chimini(1999) report that, in macroglial cells in vitro, CNTFis most prominent in the cells' nuclei. The presentevidence that CNTF is strongly expressed by RPE cellsis also novel.

In the present results, two differences in thedistributions of CNTF and bFGF seemed striking.First, in macroglial and RPE cells bFGF was prominentin nuclei and CNTF in the cytoplasm of somas, andthe difference persisted in stressed retina. Thisdifference was unexpected and suggests that the twofactors may act by distinct mechanisms. Second, bFGFbut not CNTF was prominent in the cytoplasm ofsome neuronal somas. In photoreceptors, whosesurvival is strongly enhanced by bFGF (LaVail et al.,1992; Steinberg, 1994) bFGF became prominent inthe cytoplasm of somas in regions of retina understress, again suggesting a distinct mechanism ofaction. Although several studies of the expression ofbFGF and CNTF receptors in the mammalian retinaare available (Janet et al., 1987; Raymond, Bartheland Rounsifer, 1992; Rakoczy et al., 1993; Lewis,Fisher and Anderson, 1996; Kirsch et al., 1997) nonetraces receptor distribution to the subcellular level.Kirsch et al. (1997) were unable to detect CNTFreceptor protein in situ, but noted that CNTF receptormRNA is strongly expressed by retinal neurones

(ganglion, amacrine and horizontal cells, but notphotoreceptors).

The bFGF which appears in neurones may begenerated in the neurones, or may be translocatedfrom the MuÈ ller cell processes which invest everyneurone (Hollander et al., 1991; Stone et al., 1995).We are currently studying the expression of bFGF

N. WALSH ET AL.

the source of neuronal bFGF.

Acknowledgements

This study was supported by Retina Australia, the Medical

Health and Medical Research Council of Australia.

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