Developmental Brain Research 132 (2001) 81–86www.elsevier.com/ locate /bres

Short communication

Bcl-2 expression during the development and degeneration ofRCS rat retinae

a,b ,*Rajesh Kumar SharmaaEye Pathology Institute, University of Copenhagen, Copenhagen DK-2100, Denmark

bDepartment of Ophthalmology, University of Tennessee Health Science Center, Memphis, TN 38163, USA

Accepted 21 August 2001

Abstract

In various hereditary retinal degenerations, including that in Royal College of Surgeons (RCS) rats, the photoreceptors ultimately dieby apoptosis. Bcl-2 is one of the genes, which regulates apoptosis and is thought to promote survival of cells. This study has investigatedthe developmental expression of Bcl-2 in RCS rat, which is a well-studied animal model for hereditary retinal degeneration. An antibodyagainst Bcl-2 was used for its immunohistochemical localization in dystrophic RCS rat retinae from postnatal (PN) days 4, 7, 13, 35, 45,70, 202 and 14 months. Results were compared with Bcl-2 localization in congenic non-dystrophic rats from PN 4, 7, 13, 44, 202 and 14months. Bcl-2 immunoreactivity in non-dystrophic retinae was already present in PN 4 retinae in the nerve fiber layer (presumably in the

¨ ¨endfeet of immature Muller cells) and in the proximal parts of certain radially aligned neuroepithelial cells / immature Muller cell radial¨processes. With increasing age the immunoreactivity in relatively more mature Muller cell radial processes spread distally towards the

outer retina and between PN 13 and 44 it reached the adult distribution. No cell bodies in the ganglion cell layer were found to beimmunoreactive. Expression of Bcl-2 immunoreactivity in dystrophic RCS rat retinae closely resembled that of non-dystrophic retinae. Noimmunoreactivity was seen in photoreceptors or retinal pigment epithelium in dystrophic or non-dystrophic retinae. In conclusion, Bcl-2expression is not altered, either in terms of its chronology or the cell type expressing it, during retinal degeneration in RCS rats. 2001Elsevier Science B.V. All rights reserved.

Theme: Disorders of the nervous system

Topic: Degenerative diseases: other

Keywords: Retinal degeneration; Differentiation; Apoptosis; Degenerative disease; RCS rat

Retinal degeneration is a common cause of blindness in death. A mutation in this gene also causes retinal degenera-the elderly human population. Although, efforts are being tion in humans [7].made to find treatment for hereditary retinal degenerations, Available evidence suggests that different stages ofthere is yet no effective therapy [14,17]. In various animal apoptosis are controlled by multiple genes, some of whichmodels of retinal degeneration, such as retinal degenera- are inducers and others inhibitors of cell death [6,9]. Fortion (rd) mouse, retinal degeneration slow (rds) mouse, instance, interleukin-1 b-converting enzyme (ICE) ismouse transgenic to rhodopsin mutation, vitiligo mouse, essential for apoptosis, whereas Bcl-2 and Bcl-X preventand in Royal College of Surgeons (RCS) rats, apoptosis is apoptosis. It has been demonstrated in transfected cellthe final pathway leading to photoreceptor death [12,20]. cultures and transgenic mouse that the expression of genesIn RCS rats, the mutation probably resides in the gene such as bcl-2 can prevent apoptosis [1,19]. Overexpressionencoding the receptor tyrosine kinase Mertk [5], which of Bcl-2 in retina is being envisioned as a possibleresults in inability of RPE cells to phagocytize the photo- treatment modality to prevent apoptosis in hereditaryreceptor outer segments, and eventually the photoreceptor retinal degeneration [2,17].

The pattern of bcl-2 expression changes during retinaldevelopment [3,16]. It has also been demonstrated thatBcl-2 is upregulated in the retina under certain pathologi-*Tel.: 11-901-448-1376; fax: 11-901-448-5028.

E-mail address: rksharma@utmem.edu (R.K. Sharma). cal conditions involving apoptosis of retinal neurons [4]. It

0165-3806/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0165-3806( 01 )00259-0

82 R.K. Sharma / Developmental Brain Research 132 (2001) 81 –86

is therefore interesting to determine how the pattern of nerve fiber layer was more intensely stained as comparedBcl-2 expression changes during the development of RCS to the earlier stage. Although certain cells bodies in therat retinae, and if photoreceptors express Bcl-2 at any stage ganglion cell layer appeared stained but under high magni-in an effort to survive. fication it was noted that this was due to overlapping of the

2 2 ¨Pink-eyed RCS rats (RCS-rdy p ) of postnatal days radial process of immature Muller cells. This differentia-¨(PN) 4 (n52), 7 (n52), 13 (n52), 35 (n52), 45 (n52), 70 tion was possible because immature Muller cell processes

(n52), 202 (n52), and 14 months (n52) and their could be followed radially, navigating around the cell¨congenic controls from PN 4 (n52), 7 (n52), 13 (n52), bodies in the ganglion cell layer. At this stage, the Muller

44 (n52), 54 (n52), 202 (n52) and 14 months (n52), cell endfeet were labeled and their radial processes werewere used in this study. In pilot studies, no differences in seen deeper into the inner nuclear layer than in the earlierBcl-2 expression were found between age-matched stage. Faint immunoreactivity could also be seen in theSprague–Dawley rats and congenic controls. The experi- distal most part of the retina at the location of outer

¨ments were conducted according to the rules set by the limiting membrane (Fig. 1B). In PN 13 retinae, thin MullerARVO resolution on animal experimentation. cell radial processes became more immunoreactive, reach-

Immunohistochemistry was performed on 4% parafor- ing up to the outer limiting membrane. No sign ofmaldehyde fixed cryosectioned retinae. The sections were photoreceptor degeneration was apparent yet (Fig. 1C). Inincubated with the Bcl-2 monoclonal antibody (clone 124; PN 35 retina, the outer nuclear layer was thin with theDako, Denmark) in a dilution of 1:40 in PBS in a humified accumulation of debris in the subretinal space. Immuno-

¨chamber overnight. Bcl-2 antibody is raised against a reactivity was found in the Muller cell endfeet and in thesynthetic peptide corresponding to amino acids 41–54 of radial processes. The radial processes at this stage ap-the human Bcl-2 protein (manufacturer’s data). This anti- peared robust and the outer limiting membrane wasbody reacts specifically with Bcl-2 oncoprotein as demon- distinctly immunoreactive (Fig. 1D). In PN 45 retina, thestrated by immunoblotting and immunoprecipitation [11]. picture was essentially the same, but the outer nuclearOptimum working concentration and incubation time for layer appeared even thinner (Fig. 1E). In PN 70 retina, thethe antibody was determined earlier in pilot experiments. outer nuclear layer was almost lost and there was some

¨After primary incubation, the slides were rinsed with PBS sprouting of the Muller cell processes into the subretinaland incubated for 30 min in peroxidase-conjugated goat space containing the outer segment debris. This sproutinganti-mouse secondary antibody (Dako) in a dilution of was mainly seen in the central retina (Fig. 1F). In PN 2021:30. Antigen–antibody complexes were detected by retina, the outer nuclear layer was completely lost; how-

¨avidin–biotin–peroxidase method with chrome enhance- ever, the immunoreactive Muller cells were still radiallyment (LSAB method; Dako ChemMateE Detection Kit, arranged (Fig. 1G). In 14-month-old retinae, the retinalDako). Adjacent sections were in addition also stained with degeneration was very advanced and the morphology of

¨haematoxylin for 2 min for the orientation. At least two the Muller cells, even in the inner nuclear layer, appeared¨slides from each specimen were stained, and in each distorted. The Muller cell processes in the debris filled

¨experiment, controls were obtained by omitting the pri- subretinal space were distinct (Fig. 1H). The Muller cellmary antibody. sprouting was more marked in the central retina as

In PN 4 retina, faint immunoreactivity was seen in the compared to the peripheral.nerve fiber layer and the certain radial processes presumab- The expression of immunoreactivity in the developing

¨ly that of the immature Muller cells as judged by their non-dystrophic retina closely resembled that in thehistological appearance (Fig. 1A). It was not possible to dystrophic RCS rat retina. At PN 4, immunoreactivity wasdifferentiate the ganglion cell axons from the endfeet of seen in the nerve fiber layer and weakly so in the proximal

¨ ¨Muller cells in the stained specimens. In PN 7 retina, the parts of the radial processes of Muller cells (Fig. 2A). In

Fig. 1. Bcl-2 immunoreactivity in developing dystrophic RCS rat retinae. (A) PN 4 retina, showing immunoreactivity in the nerve fiber layer and in the¨ ¨radial processes of the Muller cells (arrows). (B) PN 7 retina, showing immunoreactivity in the nerve fiber layer, Muller cells endfeet and the radial

¨processes (arrows). The outer limiting membrane is also immunoreactive (arrowheads). (C) PN 13 retina, showing intense immunoreactivity in the Mullercell processes surrounding the neurons in the ganglion cell layer and in the radial processes (arrows), which reach up to the outer limiting membrane

¨(arrowheads). (D) PN 35 retina showing thinning of the outer nuclear layer. The immunoreactivity is seen in the Muller cell endfeet, their radial processes¨(arrows) and the outer limiting membrane (arrowheads). (E) At PN 45, the picture is essentially the same as in PN 35. Muller cell radial processes (arrows)

¨are thickened. (F) At PN 70, the degeneration of the outer nuclear layer is advanced. The Muller cell radial processes are intensely immunoreactive(arrows) and these processes extend and sprout (white arrow) into the debris-filled subretinal space (Db). (G) At PN 202, the outer nuclear layer has totally

¨degenerated and the stout radial processes of the Muller cells are intensely stained (arrows). (H) Fourteen months after birth, the RCS rat retina is¨ ¨extensively degenerated. Disorganization of the Muller cells can be seen in the inner retinal layers. The radial processes of Muller cells extend into the

debris filled subretinal space (Db) and sprout there (white arrows). OS, outer segments; OLM, outer limiting membrane; ONL, outer nuclear layer; OPL,outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NFL, nerve fiber layer; arrows show radial processes

¨of the Muller cells, arrowheads show immunoreactive outer limiting membrane, asterisks show non-immunoreactive soma in the ganglion cell layer, white¨arrows mark the Muller cell processes in debris filled subretinal space.

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Fig. 2. Bcl-2 immunoreactivity in the developing non-dystrophic rat retinae. (A) PN 4 retina, showing faint immunoreactivity in the proximal part if the¨radial processes (arrows), and the endfeet of the Muller cells. No immunoreactivity is noted in the cell soma (asterisks) of the ganglion cell layer. (B) At

PN 7, immunoreactivity is still confined to the radial processes (arrows) and the endfeet plate. A part of the inner plexiform layer and region around it is¨enlarged in the inset to show that the cell soma in the ganglion cell layer are non-reactive. (C) At PN 13, the radial processes of the Muller cells (arrows)

have become immunoreactive beyond the outer plexiform layer and some immunoreactivity is visible in the outer limiting membrane (arrowheads). (D) At¨PN 44, the immunoreactivity has attained adult pattern. Muller cell radial processes are even more robustly stained (arrows) and the immunoreactivity in

the outer limiting membrane is more intense (arrowheads). (E) The immunoreactivity pattern in PN 202 retina is the same as in PN 44. (F).Immunoreactivity in a 14-month-old rat retina is unchanged as compared to adult stages. OS, outer segments, OLM, outer limiting membrane; ONL, outernuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NFL, nerve fiber layer; arrows

¨show radial processes of the Muller cells, arrowheads show immunoreactive outer limiting membrane, asterisks show non-immunoreactive soma in theganglion cell layer.

R.K. Sharma / Developmental Brain Research 132 (2001) 81 –86 85

the nerve fiber layer, distinction could not be made neurons may respond by synthesizing their own some of¨between the Muller cell endfeet plate and the ganglion cell the needed gene products.

axons. At PN 7, the immunoreactivity in the nerve fiber Retinal pigment epithelium is the most important site¨layer and in the radial processes of the Muller cells became affected by the mutation in RCS retinae and under certain

more prominent (Fig. 2B). In addition, some immuno- conditions, RPE cells are capable of up-regulating Bcl-2reactivity could be seen in the outer limiting membrane. [10]. However, in this study there was no evidence that theThe nonreactive cell bodies of the cells in the ganglion cell RPE cells in either the RCS rat retinae or the controllayer could easily be seen under high magnification (Fig. expressed detectable amount of Bcl-2.2B inset) as well as in the sections counterstained with There was no expression of Bcl-2 in the photoreceptor

¨haematoxylin. At PN 13 the Muller cell radial processes cells of either dystrophic or non-dystrophic retinae, sug-became more distinct and extended beyond the outer gesting that Bcl-2 is neither present nor up-regulated inplexiform layer where they were relatively less distinguish- these cells to the extent that this could be detected by

¨able (Fig. 2C). At PN 44, the Muller cell radial processes immunohistochemistry. When Bcl-2 is overexpressed inin the outer retina also became distinct. Furthermore, the the photoreceptors using virus-mediated delivery ofouter limiting membrane was more robustly immuno- rhodopsin-promoted bcl-2, these cells are rescued fromreactive (Fig. 2D). No immunoreactivity was seen in the degeneration [1]. This suggests that even though photo-perikarya of the cells of the ganglion cell layer in any receptor cells do not contain detectable amounts of Bcl-2stages. At PN 202 (Fig. 2E) and in 14-month-old (Fig. 2F) they may have the mechanism by which Bcl-2 can preventretina, the pattern of immunoreactivity remained un- the apoptotic cell death in these cells.changed.

The expression of Bcl-2 during the development of thenon-dystrophic control retina is in accord with previous Acknowledgementsreports [3,16]. However, because of intense immuno-reactivity it was difficult to differentiate if the immuno- Author wishes to thank Professor Jan Ulrik Prause forreactivity in the nerve fiber layer was only because of ¨valuable comments and Dr. Ozlen Konu for reading the

¨Muller cell endfeet or in addition ganglion cell axons were ¨proof. This study was financed by Ojenfonden and Civil-also involved. In dystrophic RCS retinae Bcl-2 immuno- ¨ingenor Lars Andersens Legat. Study was also funded byreactivity was comparable to that in the non-dystrophic grants from the National Eye Institute (EYO-1655 andcontrol retinae. As the retina becomes thinner due to the EY-13080), Research to Prevent Blindness, Internationalloss of outer nuclear layer in the advanced stages of retinal Retina Research Foundation, UT Memphis Neuroscience

¨degeneration, the Muller cells adapt their shape [18,15]. Center and Gerwin scholarship.¨Despite the change in the Muller cell morphology and

extensive loss of the photoreceptor cells, the localization of¨Bcl-2 with in the Muller cells was comparable to that of

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