Nestin expression in Müller glial cells in postnatal rat retina and its upregulation following optic nerve transection

Neuroscience 143 (2006) 117–127

NESTIN EXPRESSION IN MÜLLER GLIAL CELLS IN POSTNATAL RAT RETINA AND ITS UPREGULATION FOLLOWING OPTIC NERVE TRANSECTION L. P. XUE,a,b J. LU,c Q. CAO,a C. KAURa AND E.-A. LINGa*

nestin positive cells existed in neonatal rat retinas (Wu et al., 2004), adult human retina and within surgically removed epiretinal membranes (Mayer et al., 2003). The possibility that nestin positive cells represent a population of progenitor cells which can differentiate to form retinal scar tissue and the possibility that rat retina has the ability of regeneration throughout its life have been considered (Wu et al., 2004; Mayer et al., 2003). This would have immense clinical implications because loss of retinal ganglion cells (RGCs) in several neurodegenerative diseases such as optic nerve injury, acute or chronic glaucoma is well documented. In a study in both differentiated and undifferentiated human fetal retina, Walcott and Provis (2003) reported that retinal progenitors and Müller glial cells expressed nestin and considered them as a single cell class. Furthermore, it has been reported recently that transitin, a nestin-related intermediate filament, is expressed by neural progenitors and can be induced in Müller glial cells in the chicken retina following intraocular injections of NMDA (Fischer and Omar, 2005). Recently, we (Xue et al., 2006) have shown that under normal circumstances, nestin immunoexpression was almost undetected in the Müller glial cells in adult rats but it could be induced in experimentally induced glaucoma. While it is unequivocal from our study that Müller glial cells express low levels of nestin in the adult retina, its expression pattern in the developing retina has remained to be elucidated. It also remains to be ascertained if the low level of nestin expression in the adult retina would be affected in acute retinal damage such as injuries to the optic nerve. Given their close spatial relation with the RGCs, it is conceivable that Müller glial cells would play a crucial role in maintaining the integrity of neurons in normal development and retinal pathologies. Hence, the characterization of the immunophenotypic profiles of Müller glial cells including their possible nestin expression during development and response to lesion to RGC degeneration in optic nerve lesion may provide clues to a better understanding of the roles of these cells in the retina. We report here the localization and expression of nestin in the rat retina beginning at birth (postnatal day 0) until maturity. The expression patterns of nestin at different time points after optic nerve transection (ONT) were also followed. We examined the relationship between the nestin-labeled retinal progenitor cells and Müller glial cells and compared this with cells labeled by glial fibrillary acidic protein (GFAP), neuronal nuclear antigen (NeuN) and glutamine synthetase (GS), an established marker for Müller glial cells. A major finding is that nestin is expressed by neural progenitors as well as

a

Department of Anatomy, Yong Loo Lin School of Medicine, Block MD 10, 4 Medical Drive, National University of Singapore, Singapore 117597

b

Zhongshan Ophthalmic Center, Sun Yat-Sen University, 54 Xianlie, South Road, Guangzhou, PR China 510060

c

Defence Medical and Environmental Research Institute, DSO National Laboratories, 27 Medical Drive, Singapore 117510

Abstract—This study examined the nestin immunoexpression and its specific cellular localization in the developing retina of rats and investigated its putative changes in an altered environment. At postnatal day 0, nestin immunoexpression was detected in radially oriented cells considered to be neural progenitors that were glutamine synthetase (GS) negative. With age, it was localized in differentiating and differentiated GS positive Müller glial cells. Nestin expression was down-regulated as maturation proceeded, so that by 12 weeks, it was almost completely diminished as confirmed also by real time–polymerase chain reaction analysis. Nestin expression along with that of glial fibrillary acidic protein (GFAP) was induced and upregulated in mature Müller glial cells following optic nerve transection. It is suggested that both nestin and GFAP may be useful biomarkers in retinal injuries. In view of their cytoskeletal nature, the marked expression of nestin and GFAP may provide a structural support for the framework of retina which would be disrupted as a result of loss of neurons in optic nerve lesion. It may also be neuronal protective taking into consideration the close spatial and functional links between Müller glial cells and the axotomized ganglion cells. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: Müller glial cells, nestin, glutamine synthetase, development, optic nerve lesion.

The nestin gene, encoding a sixth class of intermediate filaments, was originally cloned from an E15 rat CNS cDNA library (Lendahl et al., 1990). Nestin protein localizes mainly in neural progenitor cells during murine CNS development (Hockfield and McKay, 1985; Lendahl et al., 1990; Craig et al., 1996). It has been widely used as a cell distinguishing marker of neural progenitors in the mammalian nervous system. Recent studies have reported that *Corresponding author. Tel: ⫹65-6874-3200; fax: ⫹65-6778-7643. E-mail address: [email protected] (E.-A. Ling). Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; GCL, ganglion cell layer; GFAP, glial fibrillary acidic protein; GS, glutamine synthetase; INL, inner nuclear layer; NeuN, neuronal nuclear antigen; NFL, nerve fiber layer; NR, neural retina; ONT, optic nerve transection; PB, phosphate buffer; PBS-TX, 0.01 M phosphate buffered saline at pH 7.4 containing 0.1% Triton X-100; PCR, polymerase chain reaction; RGC, retinal ganglion cell; RPE, retinal pigment epithelium.

0306-4522/06$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.07.044

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differentiating and differentiated Müller glial cells as determined by GS labeling. Furthermore, nestin can be induced and upregulated in Müller glial cells by acute retinal damage suggesting that it may be useful marker in retinal injuries.

EXPERIMENTAL PROCEDURES A total of 36 Wistar rats, aged 1 day (P0) and 1, 2, 3, 4 and 12w (n⫽6 for each age group) were used for developmental study. In addition, 18 adult rats aged 12w were used for ONT.

Surgical procedures Eighteen adult female rats aged 12w (weighing 220 –270 g) were deeply anesthetized with an i.p. injection of Nembutal (100 mg/ kg). The left optic nerve was transected intraorbitally according to the Sarup et al. (2004) method. Briefly, the anesthetized rats were placed on a stereotactic apparatus. Skin incision along with superficial dissection was then carried out avoiding the supraorbital vein. The lacrimal gland was partially dissected where necessary and the superior extraocular muscles were retracted. The optic nerve was exposed and the dura mater cut longitudinally. Complete ONT was performed at an identical location that was 3 mm away from the globe for all rats. Care was taken to avoid injury to the ophthalmic artery and traction to the optic nerve. The vascular circulation of the retina was assessed by the red reflex from the eye. Skin was sutured and erythromycin eye ointment was applied to the skin and eye. The right eye was sham operated and used as control. Six rats each were killed at 1d and 1w postoperation for immunofluorescence. Three rats each were killed for extraction of total RNA and real time–polymerase chain reaction (PCR) analysis. In the handling and care of all animals, the International Guiding Principles for animals research, as stipulated by the Council for International Organizations of Medical Sciences (CIOMS) (1985) and as adopted by the Laboratory Animal Centre, National University of Singapore, were followed. All efforts were made to minimize the number of rats used and their suffering.

Perfusion and tissue preparations for immunofluorescence Following deep anesthesia, the rats were first perfused with Ringer’s solution until the lungs and liver were cleared of blood and then followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). After perfusion, the eyeballs were removed and immediately immersed in the same fixative for 2– 4 h before they were transferred into 0.1 M PB containing 20% sucrose and kept overnight at 4 °C. Frozen sagittal sections of the eye were cut at 20 ␮m thickness and mounted on chrome-alum gelatin-coated slides. Sections were air-dried and stored at ⫺20 °C until use.

RNA extraction and RT-PCR analysis Total RNA was extracted from the retina from the experimental and control rats. Using the RNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer’s instructions and quantified spectrophotometrically at 260 nm. Reverse transcription reactions were performed using reverse transcription system (Promega, Madison, WI, USA). Briefly, each 20 ␮l cDNA synthesis mixture contained 1 ␮g total RNA, 4 ␮l MgCl2 (25 mM), 2 ␮l reverse transcription 10⫻ buffer, 2 ␮l of 10 mM deoxynucleotide triphosphates (dNTP), 0.5 ␮l of recombinant RNasin ribonuclease inhibitor, 15 U of AMV reverse transcriptase, and 0.5 ␮g oligo(dT)15 primers. The resultant cDNA was diluted 10 times in double-distilled H2O and aliquoted, kept at ⫺20 °C for real time-PCR analysis. Gene sequences for primer design were obtained from the National Centre for Biotechnology Information’s GeneBank. Primer pairs for nestin, GS and GFAP were designed using the

program primer design (primer 3 software version 1.0). The forward and reverse primer sequences of CNPase were 5=-caaccacaggagtgggaact-3= and 5=-tctggcattgactgagcaac-3=; GS primers were 5=-aagctggtgttctgcgaagt-3= and 5=-gccataagcctgtcagctc-3=; GFAP primers were 5=-gaagaaaaccgcatcaccat-3= and 5=-gcacacctcacatcacatcc-3=, respectively. Real time-PCR was performed using a LightCycler (Roche Diagnostics, Indianapolis, IN, USA) and individual PCR reactions were carried out in glass Light Cycler capillaries (Roche) according to the manufacturer’s instructions. Briefly, the PCR reaction was carried out in a 20 ␮l final volume containing the following: 4 ␮l SYBGreen I master mix (Roche Applied Science), 2 ␮l 10 ␮M forward primer and reverse primer; and 1.0 ␮l diluted cDNA and 11 ␮l 0.1% diethylpyrocarbonate-treated water (DEPC H2O). After an initial denaturation step at 95 °C for 10 min, temperature cycling was initiated. Each cycle consisted of denaturation at 95 °C for 5 s, annealing at 61 °C for 5 s (nestin and GS) and at 60 °C for 5 s (GFAP), and elongation at 72 °C for 4 s. A total of 35 cycles were performed. Rat GAPDH was amplified as the control for normalizing the quantities of transcripts of the above gene. The expression difference for nestin, GS, and GFAP genes in the retina of different development stages and after ONT was calculated by normalizing with GAPDH gene expression according to 2⫺⌬⌬T method (Livak and Schmittgen, 2001).

Müller glial cells culture One week old rats were used for Müller glial cells culture according to the modified Hicks’s method which offers a simple rapid reproducible method for obtaining large quantities of purified Müller cells that do not dedifferentiate or express high levels of GFAP (Hicks and Courtois, 1990). The rats were killed instantly by cervical dislocation and the eyes were rapidly enucleated into Dulbecco’s modified Eagle’s medium (DMEM) containing 2 mM glutamine and 1:1000 penicillin/streptomycin, and stored overnight at 37 °C in the dark. Eyes were treated according to the protocol of Edwards (Edwards, 1981). Briefly, intact globes were incubated in 0.1% trypsin at 37 °C for 60 min. They were then placed in a Petri dish containing DMEM supplemented with 10% fetal calf serum (FCS). After this, the retinas were removed, either mechanically dissociated with a sterile Pasteur pipette into small aggregates or chopped into 1 mm fragments and seeded into 10 cm Falcon culture dishes with six to eight tissue specimens per dish. The medium was left unchanged for 48 h, and then the retinal aggregates and debris were removed by forcibly pipetting medium onto the dish. This procedure was repeated three to five times to dislodge all aggregates resulting in a purified flat cell population. The cells on dish surface proliferated rapidly, becoming fully confluent within 4 – 8 days after initial appearance. At this time, cells could be easily passaged after rinsing twice with PBS followed by a brief incubation in PBS containing 0.1% trypsin. Finally the cells were resuspended and seeded in fresh DMEM⫹10% FCS and the medium was changed every 3– 4 days. All cultures were maintained at 37 °C in a 5% CO2/95% air atmosphere in a humidified incubator. Antiserum to GS considered a reliable marker for Müller glial cells was used for identification of cells.

Immunohistochemistry Mounted sections or cultured Müller glial cells on coverslips were washed for 20 min in 0.01 M phosphate buffered saline (PBS) at pH 7.4 containing 0.1% Triton X-100 (PBS-TX) solution and then blocked by 2% normal goat serum for 1 h. Double immunofluorescence labeling was carried out with primary antibodies listed in Table 1 overnight at room temperature. After incubation with the respective primary antibodies, tissue sections were washed three times with PBS-TX for about 15 min. The secondary antibodies used were: anti-mouse IgG conjugated with Cy3 (1:200; Chemicon International; Temecula, CA, USA) and anti-rabbit IgG conjugated with FITC (1: 200; Sigma, St. Louis, MO, USA). Incubation in the secondary antibodies was carried out in the dark at room temperature for 1 h. After

L. Xue et al. / Neuroscience 143 (2006) 117–127 Table 1. Antibodies and source of supply and dilution used Antibodies

Source

Host

Dilution

Nestin GFAP GS NeuN

Chemicon Chemicon Chemicon Chemicon

Mouse Mouse Rabbit Mouse

1:200 1:800 1:1000 1:200

further rinsing the sections were mounted with Dako fluorescent mounting medium (Dako, Carpenteria, CA, USA) and covered with coverslips. Negative controls were performed routinely by incubating the sections in normal buffered serum instead of the primary antibody. Sections were examined in an Olympus confocal laser scanning microscope (FV 1000; Olympus, Tokyo, Japan). The sections were finally dehydrated in graded concentrations of alcohol and mounted in permount.

Cell counting Retinal sections from rats killed at 1d and 1w after ONT immunostained for NeuN were used for cell enumeration (n⫽3 at each

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time interval). Only NeuN positive cells in ganglion cell layers (GCL) were identified as RGCs. Three retinal sections prepared through the level of the optic disc were used. A total of three retinal areas each outlined by an ocular graticule, measuring 0.03 mm2 were sampled equidistantly across the GCL as described previously (Zeng et al., 2000). Enumeration of immunoreactive cells was then performed with an Olympus microscope at a magnification of 400⫻. For each eye, a total of nine fields were scrutinized and the values obtained were averaged. A Bonferroni test in the ANOVA was used to evaluate significant changes statistically; P⬍0.01 was considered significant.

RESULTS Expression of nestin, NeuN, GFAP and GS during retinal development At P0 (Fig. 1a–i), the retina showed two distinct layers: the outer retinal pigment epithelium (RPE) and the inner neural retina (NR) (Fig. 1a). Nestin immunoreactivity was distributed throughout the retina being localized primarily in elon-

Fig. 1. (a–i) At P0, the retina is differentiated into two distinct layers: the outer RPE that is intensely immunoreactive for GS, and the inner NR that lacks GS staining (a). At this time point, nestin immunoreactivity is localized throughout the retina being localized primarily in elongated cells (arrows) radiating from the inner to the outer limits of the NR (b). The nestin immunoreactivity appears to be more intense nearer to the inner limiting membrane (b, c). NeuN positive neurons (arrow) are distributed in the inner layer of retina (e), the presumptive GCL and NFL where GFAP positive astrocytes are known to exist (arrows, h). Note the lack of colocalization between GS and NeuN (d–f). Colocalization, however, is apparent between GFAP cells with GS (arrows in g–i). Scale bar⫽100 ␮m in f (applies to d–f). Scale bar⫽50 ␮m in i (applies to a– c, g–i).

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gated retinal progenitor cells radiating from the inner to the outer limits of the NR (Fig. 1b). Nestin positive cells and their slender processes spanned the retina reaching as far as the RPE that was intensely stained with GS (Fig. 1c). Nestin immunoreactivity appeared to be more intense in stout processes in palisade that were anchored to the inner limiting membrane (Fig. 1b, c). NeuN positive neurons were restricted to the inner zone of the retina and were not colocalized with GS immunostaining (Fig. 1d–f), where a

variable number of GS and GFAP positive cellular elements were also present. The GFAP positive cells identified as astrocytes appeared to overlap with GS immunostaining (Fig. 1g–i). At 1w (Fig. 2a– k), nestin was detected throughout the retina but preferentially in the inner layer of the NR (Fig. 2b). Meanwhile, many elongated GS positive cells whose somata were localized in the middle of the NR corresponding to the inner nuclear layer (INL) were observed (Fig. 2a).

Fig. 2. (a– k) At 1w, a large number of GS positive cells considered to be Müller glial cells and whose cell bodies lie in the INL are identified (a). Nestin immunoreactivity is detected across the layers being more intensely stained in the inner layer of the NR (b). Müller glial cells are double labeled for nestin and GS (arrows in a, b and c and also d). NeuN positive cells are comparable to that of P0 (f). There was no evidence of cells that are double labeled for NeuN and GS (e– h). GFAP immunoreactivity is localized in astrocytes in GCL/NF. Colocalization is observed between GS and GFAP in some astrocytes (i– k). Scale bar⫽20 ␮m in h (applies to d). Scale bar⫽50 ␮m in k (applies to a– c, e– g, i– k).

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The radially oriented GS cells were identified as Müller glial cells whose outer and inner processes extended to the outer and inner limiting membranes, respectively. Double labeling study revealed that the nestin positive cells were colabeled for GS (Fig. 2c, d), the immunoreactivity being more marked at the inner processes and end-feet of Müller glial cells when compared with the somata (Fig. 2c, d). Like in P0 rats, NeuN positive cells appeared as a distinct row of cells; none, however, were colabeled with GS (Fig. 2e– h). GFAP positive cells were confined mainly to the inner layer of the retina where astrocytes are known to exist (Fig. 2j, k). As in P0 rats, GFAP immunolabeling superimposed with GS (Fig. 2i– k). At 2w (Fig. 3a– h), nestin immunoreactivity was distinctly localized in Müller glial cells (Fig. 3b– d). They were colabeled for GS both at their somata and long slender processes in parallel arrays (Fig. 3b– d) thus confirming their identification. Besides the RGCs in the GCL, some NeuN positive bipolar cells were identified in INL (Fig. 3e). The expression of GFAP was comparable to that observed at 1w being localized mainly in astrocytes in the GCL/nerve fiber layer (NFL) (Fig. 3g). As in P0 and 1w, some GFAP positive astrocytes showed colocalization of GS (Fig. 3f– h). A noteworthy feature at this age group was the localization of nestin immunoreactivity associated with some blood vessels (Fig. 3b).

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At 3w, nestin immunoreactivity remained to be localized in Müller glial cells that were colabeled with GS (Fig. 4a– e). It was progressively reduced with advancing age. The diminution of nestin labeling was evident at 4w (Fig. 4f–i) and that by 12w, the immunolabeling was almost undetected (Fig. 4k–m). A salient feature at 3w and 4w was that nestin protein tended to accumulate at the expanded end-feet of Müller glial cells resting on the inner limiting membrane (Fig. 4c, h). In rats older than 3w, NeuN immunoreactivity in GCL contained more immunolabeled neurons (data not shown). Like in younger rats, GFAP immunolabeling was confined to astrocytes in GCL/NFL (data not shown). Expression of nestin, GS, and GFAP mRNA in retinal development Real time-PCR analysis of retina in development showed that a clear band was detected for nestin mRNA at 220 bp, GS mRNA at 234 bp, GFAP mRNA at 190 bp (data not shown). Rat GAPDH was amplified as the control. Nestin mRNA that was vigorously expressed at birth (P0) was progressively and significantly decreased in the developing retina with advancing age (Fig. 5); it was barely detected at 12w. GS mRNA expression was increased from P0 till 3w, peaking at this age; hereafter, the GS expression was markedly decreased (Fig. 5). GFAP mRNA ex-

Fig. 3. (a– h) At 2w, nestin immunoreactivity is strongly expressed by Müller glial cells colabeled for GS (arrows in a– d, (d) is enlarged view of boxed area in (c)). (b) NeuN immunolabeling in GCL is comparable to that at 1w except that immunolabeled neurons are now observed in the INL (e). GFAP immunolabeling that is confined to GCL/NFL is comparable to that of 1w (g); there is overlapping (arrows) between GFAP and GS in some cells (f– h). Arrowheads in b– d, blood vessels. Scale bar⫽20 ␮m in d. Scale bar⫽50 ␮m in h (applies to a– c, e– h).

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Fig. 4. (a–m) GS and nestin coexpression at 3w (a– e), 4w (f–j) and 12w (k–m). From 3w till 4w, nestin positive cells are colabeled with GS (b– e, g–j), but with age, the nestin immunoreactivity is progressively diminished. It is markedly attenuated and is barely detected at 12w (l). At 3w and 4w, the immunoreactivity appears to be more intensely stained at the expanded end-feet of Müller glial cells (c, h). Scale bar⫽50 ␮m in m (applies to a, b, d, f, g, i, k–m). Scale bar⫽20 ␮m in j (applies to c, e, h, j).

pression was very low initially but rose after 3w, peaking at 4w and declined thereafter (Fig. 5). Expression of nestin and GS after ONT As described above, nestin expression in the retina was greatly reduced and was negligible in Müller glial cells at 12. In the light of this, we sought to determine whether nestin expression would be induced at this age group following axotomy of the RGCs because both cell types are closely related spatially and functionally. At 1d after ONT (Fig. 6a– c), nestin expression was moderately increased in the GCL and NFL. Nestin immunoreactivity was localized in Müller glial cells spanning the different layers of the retina (Fig. 6b– c). Nestin immunoreactivity was markedly induced at 1w in Müller glial cells colabeled with GS (Fig. 6d–f). While nestin immunoreactivity was observed throughout Müller glial cell somata and processes, it appeared to be more intense at or near the end-feet (Fig. 6e).

Expression of GFAP after ONT In the sham-operated retina, antibody directed against GFAP specifically labeled astrocytes in the GCL and NFL but not Müller glial cells. Following ONT, many GFAP immunoreactive cells and processes appeared to traverse through all the retinal layers reaching as far as the photoreceptor layer. The increase and induced GFAP immunoreactivity was evident at 1d after ONT (Fig. 6h) and was further augmented at 1w (Fig. 6k). The GFAP immunoreactivity in the long extending processes was superimposed with GS staining confirming that they were Müller glial cells (Fig. 6g–l). Expression of nestin, GS, GFAP mRNA in retina after ONT At 1d and 1w after nerve transection (ONT), the nestin, GS and GFAP mRNA level was significantly increased when compared with the controls (Fig. 5).

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Fig. 5. Bar graphs in real time-PCR analysis showing nestin expression in developing rat retina and after optic nerve transaction (upper panel); GS expression in developing rat retina and after optic nerve transaction (middle panel); GFAP expression in developing rat retina and after optic nerve transaction (lower panel). Note the progressive diminution of nestin mRNA expression with age (upper panel). It is highly expressed at P0 and is negligible at 12w. In rats whose optic nerve is transected, nestin mRNA expression is upregulated being very conspicuous at 1w after operation. GS mRNA expression is hardly detected at birth (PO), but is enhanced with age peaking at 3w and reduces thereafter (middle panel). GS mRNA expression is also upregulated after optic nerve transaction. GFAP mRNA expression increases with age peaking at 4w but declines sharply thereafter (lower panel). It is markedly enhanced after ONT.

Expression of NeuN after ONT In sham-operated retina, monoclonal NeuN antibody labeled the neurons in the GCL and INL. NeuN immunore-

activity was localized in the neuronal somata and some processes (Fig. 7a). The number of NeuN-stained neurons in the GCL appeared to be reduced at 1d after ONT (data

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Fig. 6. (a–f) Nestin immunoreactivity is markedly induced in Müller glial cells at 1d (b) and 1w (e) after ONT. All nestin positive cells are colabeled with GS (arrows in a–f). Following ONT at 1d and 1w, GFAP immunostaining is enhanced. Many GFAP immunoreactive cells and processes span through all layers of retina with some processes extending as far as the photoreceptor layer (arrows in h, k). GFAP immunoreactivity is most intense in the vicinity of ganglion cells where astrocytes and end-feet of Müller glial cells are intermingled. Scale bar⫽50 ␮m (applies to a–l).

not shown) so that by 1w (Fig. 7b) it is estimated that the loss of NeuN positive cells was at least 40% when com-

pared with the corresponding sham-operated control eye (Fig. 7c).

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Fig. 7. (a– c) In sham-operated retina, neurons in the GCL are NeuN positive; some neurons in the INL are also stained (a). NeuN immunoreactivity is mainly localized in the neuronal somata and processes. The number of NeuN-stained neurons in the GCL is noticeably reduced after ONT (b), so that by 1w there is loss of as much as 40% of the NeuN-positive cells compared with the corresponding control (c). Each column in (c) represents the number of positive RGCs in 0.173 mm of retinal tissue (mean⫾S.D.). (** P⬍0.01). Scale bar⫽100 ␮m in a; scale bar⫽50 ␮m in b.

Immunocytochemical characterization of cultured Müller glial cells In order to ascertain whether nestin is expressed in Müller glial cells in vitro, the cells were double labeled for GS and nestin. A 1:1 ratio was noted between GS and nestin positive Müller glial cells (Fig. 8a– c). GS immunoreactivity was localized in both the cytoplasm and nucleus (Fig. 8a), whereas nestin immunolabeling was confined to the cytoplasm (Fig. 8b).

DISCUSSION Müller glial cells are a specialized type of glia whose somata lie in the middle of the INL, while their processes

span all layers of the retina in the adult. Tritiated thymidine studies suggest that Müller glia are the last cells born in the retina (Prada et al., 1989a,b), although several authors have reported that Müller glia are present in the retina from a much earlier stage of development (Derouiche and Rauen, 1995). The present results have shown that Müller glial cells as confirmed with their specific marker, GS, were first identified at 1w of age. On the other hand, bipolar neurons in the INL as recognized by their staining with NeuN occurred at 2w indicating that they are formed after the development of Müller glial cells. Müller glial cells are considered analogous to the radial glia of the cortex. Both cell types have radial processes spanning the thickness of the cortex/retina and both play a role in guiding newly born

Fig. 8. (a– c) Müller glial cells in culture coexpress nestin and GS. Scale bar⫽20 ␮m in c (applies to a– c).

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neurons to their appropriate layers (Lendahl et al., 1990; Messam et al., 2000). However, radial glia are present only during development before transforming into astrocytes (Voigt, 1989; Culican et al., 1990), while Müller glial cells persist through adulthood. Nestin is an intermediate filament protein reported in a number of recent studies to be a marker of neural progenitor cells; it is not expressed by differentiated neurons (Hatten, 1990, 1999). Nestin positive cells in the retina are regarded as progenitor cells (Walcott and Provis, 2003). Present results showed that nestin was colocalized with GS in elongated and slender cells from 1w till 7w thus confirming that they are differentiating or differentiated Müller glial cells. It has been reported that retinal differentiation in rats was complete at 2w after birth (Wu et al., 2004). Hence, the localization of nestin in GS labeled cells at 2w or older would be considered as differentiated Müller glial cells. Our findings are therefore consistent with the view that Müller glial cells express nestin in both differentiated and undifferentiated human fetal retina (Walcott and Provis, 2003). The present study in postnatal retina has revealed that nestin immunoreactivity in Müller glial cells was intense between 1 and 3w but was progressively attenuated with age so that by 12w, it was barely undetected. This suggests that fully mature Müller glial cells in older rats do not express detectable levels of nestin at the protein level. Although progenitor cells exist in the mature mammalian retina, they are mainly located in the pigmented ciliary bodies and the number is very low (Ahmad et al., 2000; Tropepe et al., 2000). We have shown that Müller glial cells in culture derived from 1w old rats express nestin and coexpressed GS. This confirms that nestin is not exclusively expressed by neural progenitor cells. Müller glial cells are the major support cells for neurons in the retina. As the major supportive glia in the retina, they are involved in both normal function and pathology of the retinal neurons (Reichenbach et al., 1993; Frisen et al., 1995). Müller glial cells can recognize a variety of neuronal signals and actively control levels of K⫹, H⫹, Na⫹ and neurotransmitters such as glutamate and GABA in the retina (Newman and Reichenbach, 1996). Damage to the optic nerve leading to the loss of ganglion cells and their axons has been demonstrated using various conventional histological methods. The present results using NeuN labeling have confirmed the loss of ganglion cells following ONT. Müller glial cell activation has been reported in response to neuronal injury, degeneration and regeneration (Eisenfeld et al., 1984; Battisti et al., 1995; Newman and Reichenbach, 1996). Under such conditions, these cells undergo reactive gliosis characterized by alteration in intermediate filament production (Kim et al., 1998). A characteristic feature is the upregulated expression of GFAP (Napper et al., 1999; Grosche et al., 1995; Lewis and Fisher, 2003). In connection with the present study, it is relevant to note that the nestin gene, in addition to its normal expression during CNS development, is reactivated in different situations of cellular stress or induced proliferation (Dahlstrand et al., 1992). It has been reported

that astrocytes express nestin after traumatic injury to the spinal cord and optic nerve (Frisen et al., 1995). Nestin expression can be induced in CNS tumors, in particular in more malignant tumors (Tohyama et al., 1992). Furthermore, cells that are removed from adult striatum and grown in primary culture express nestin (Reynolds and Weiss, 1992). Taken together, it seems apparent that cells originally derived from a nestin expressing population can resume their expression when subjected to various stimuli. Arising from our finding, it is suggested that nestin is a potentially useful marker for various retinal pathogenic conditions. Firstly, nestin is an easily recognizable cytoskeletal network. Secondly, induction and upregulation of nestin expression are rapid in onset within 1 day after injury. The significance of induced nestin expression in injury is uncertain but it is speculated that it may reflect a metabolic change of the cells in reaction to the axotomy of the ganglion cells to which they are closely associated. In view of its cytoskeletal nature, its increased production following ONT would provide a stronger rigidity for Müller glial cells and strengthen the retinal tissue that would be disrupted due to massive neuronal loss. With enhanced intermediate protein filaments, nestin and GFAP and their unique configuration, Müller glial cells would help maintain the retinal structural integrity in various retinal pathologies, among many other functions. Little is yet known about the factors controlling the nestin induction, but it is possible that the induced expression after injury is at least in part caused by an increase in certain locally derived factors, such as nerve growth factor (NGF) (Drapeau et al., 2005), basic fibroblast growth factor (bFGF) (Fischer and Omar, 2005), insulinlike growth factor (IGF) (Hodge et al., 2004) and ciliary neurotrophic factor (CNTF) (Zahir et al., 2005).

CONCLUSION In conclusion, the present results have shown that nestin is expressed in differentiating and differentiated Müller glial cells but it is down-regulated with age. In optic nerve lesion, however, nestin expression is upregulated notably in their basal processes in the GCL/NFL. Concomitantly, GFAP expression is markedly enhanced as has been reported previously by others (Chen and Weber, 2002). By virtue of their close spatial relation with the somata of ganglion cells and together with the local astrocytes, it is suggested that the reactive changes of Müller glial cells may be elicited by factors derived from the axotomized ganglion cells or microglia that are fully activated in retinal pathologies (Wang et al., 2000; Zeng et al., 2000). Induced and increased nestin expression coupled with that of GFAP is additional cytoskeletal machinery in Müller glial cells for strengthening the structural support of retinal tissue especially in injuries. Acknowledgments—This study was partially supported by a research grant from R-181-000-086-592 (DDSO-NUS) Agreement No. DSOCLO 5034, National University of Singapore.

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(Accepted 27 July 2006) (Available online 1 September 2006)