nestin expression in the retina of rats with inherited retinal degeneration

Q2

at SciVerse ScienceDirect

Experimental Eye Research xxx (2013) 1e9

12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455

YEXER6098_proof ■ 13 February 2013 ■ 1/9

Contents lists available

Experimental Eye Research

journal homepage: www.elsevier .com/locate/yexer

56575859606162636465666768

Nestin expression in the retina of rats with inherited retinal degeneration

F. Valamanesh a,b, J. Monnin c, N. Morand-Villeneuve d, G. Michel e, Z. Muraf f, S. Miloudi a,D. Chemouni a, J.C. Jeanny a, C. Versaux-Botteri a,e,*a INSERM U872, Equipe 17, Physiopathologie des maladies oculaires, innovations thérapeutiques, 15, rue de l’Ecole de Médecine, 75006 Paris, Franceb Fondation Rothschild, 29, rue Manin, 75019 Paris, FrancecHôpital Saint-Jacques, 2, Place Saint-Jacques, 25000 Besancon, Franced EA 3922, SMP, Hauts de Chazal, 25000 Besancon, FranceeUniversité de Franche Comté, 25000 Besancon, Francef INSERM U872, Equipe 18, Résistance et survie des cellules tumorales, 15, rue de l’Ecole de Médecine, 75006 Paris, France

6970717273747576777879808182

a r t i c l e i n f o

Article history:Received 10 September 2012Accepted in revised form 25 January 2013Available online xxx

Keywords:nestinMüller cellsradial gliaRCS ratimmunohistochemistryinherited retinal dystrophyWestern blottingqPCR

* Corresponding author. CRC, INSERM U872, Équimaladies oculaires, innovations thérapeutiques, 15,75006 Paris, France. Tel.: þ33 140467847; fax: þ33 1

E-mail addresses: [email protected] (C. Versaux-Botteri).

0014-4835/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.exer.2013.01.013

Please cite this article in press as: ValamanesEye Research (2013), http://dx.doi.org/10.10

83848586878889909192

a b s t r a c t

Nestin is found in radial glia and neuronal/glial progenitor cells during retinal development, and isre-expressed after acute damage in the retina of adult mammals. We have investigated nestin expressionin the retina of the Royal College of Surgeons (RCS) rat model of human inherited blindness, Retinitispigmentosa (RP). During the first postnatal week, nestin immunoreactivity was located in elongatedprocesses resembling radial glia in both control and dystrophic animals. During the second postnatalweek, the density of nestin immunoreactive radial processes decreased progressively starting in theouter retina. At postnatal day 20 (PNd20), Nestin immunoreactive radial processes were no longervisible, with immunoreactivity restricted to structures resembling Müller end-feet and/or astrocyteslocated in the ganglion cell layer (GCL) in both control and dystrophic rats. These morphological resultswere confirmed by Western blotting and qPCR analysis. The level of nestin remained low in controlanimals at different time points up to 1 year, but we observed a re-expression of this protein from PNd30in the dystrophic animals. The morphology of cells re-expressing nestin resembled that of radial glia and/or Muller cells, but co-localization of nestin and glutamine synthetase (GS: a marker of mature Müllercells) was only partial. Interestingly, whereas Western blot analysis confirmed the increase in proteinlevels from PNd30 onwards, mRNA levels remained low in dystrophic rats. Additional studies demon-strated that the discrepancy between protein and mRNA contents could be due to a dysfunction inproteasome activity as often observed in neurodegenerative pathologies. In conclusion, because of itslocalization in astrocytes and in radial processes resembling radial glia in the pathologic adult retina,nestin may be involved in mechanisms such as cell migration, generation of new neurons or glial cellsand/or in retinal (re)modeling in dystrophic adult animals. The lack of concomitant up-regulation ofmRNAs in adult dystrophic animals suggests that the pathology could lead to transcriptional and/ormetabolic changes involving the stabilization of the half-life and/or dysregulation of degradation pro-cesses of nestin protein.

� 2013 Elsevier Ltd. All rights reserved.
9394 95 96979899
100101

1. Introduction

During development and throughout life many organs containprogenitor cells that can (re)generate their tissues. These progeni-tor cells have been shown to contain nestin, a type VI-intermediate

Q1

pe 17, Physiopathologie desrue de l’Ecole de Médecine,40467855.omte.fr, claudine.botteri@

All rights reserved.

h, F., et al., Nestin expression16/j.exer.2013.01.013

102103104105106107108

filament protein. Identified in 1985 by Hockfield and McKay, nestin(for neuroepithelial stem cell protein) is considered as a reliablemarker of mitotically active stem cells and progenitor cells. Firstdescribed in the developing central nervous system (CNS), nestin isnow known to be expressed in various proliferating tissues, such asheart muscle, skeletal muscle, proliferative endothelial cells, andteeth (Lendahl et al., 1990; Sejersen and Lendahl, 1993; Kachinskyet al., 1995; Terling et al., 1995; Suzuki et al., 2010). The retina, asa part of the CNS, was thought to lack regenerative capacity in adultmammals in spite of the existence of a quiescent population of stemcells at the ciliary margin (Ahmad et al., 2000; Tropepe et al., 2000).However, nestin which is expressed in fetal retinal cells is re-

in the retina of rats with inherited retinal degeneration, Experimental

109110

mailto:[email protected]



www.sciencedirect.com/science/journal/00144835

http://www.elsevier.com/locate/yexer

http://dx.doi.org/10.1016/j.exer.2013.01.013



F. Valamanesh et al. / Experimental Eye Research xxx (2013) 1e92

111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172173174175

176177178179180181182183184185186187188189190191192193194195196197198199200201202203204205206207208209210211212213214215216217218219220221222223224225226227228229230231232233234235236237238239240


expressed in cells resembling Müller cells in adult retina after anacute injury (Walcott and Provis, 2003). Numerous studies haveemphasized the capacity of Müller cells, the predominant retinalglial cells, to express nestin in response to different acute damageparadigms such as induced glaucoma (Xue et al., 2006a), opticnerve transection (Xue et al., 2006b), laser injury (Kohno et al.,2006), kainate-induced neurotoxicity (Chang et al., 2007), N-methyl-D-aspartate (NMDA) induced neurotoxicity (Karl et al.,2008), N-methyl-N-nitrosourea (MNU) treatment (Wan et al.,2008) or experimental retinal detachment (Luna et al., 2010).Recently, Goel and Dhingra (2012) have demonstrated the presenceof this protein in retinal Müller cells in rd1 mice. These animalsdevelop an inherited degenerative retinal pathology resemblinghuman Retinitis pigmentosa (RP)e a disease leading to blindness byphotoreceptor degeneration. All these works hypothesize thatMüller cells are able to re-differentiate into retinal neurons aftera neurodegenerative disease.

In this present study, we have examined the temporal patternand cellular localization of nestin (protein andmRNAs) in the retinaof a rat model exhibiting neurodegenerative inherited disease.Experiments were carried out on dystrophic Royal College of Sur-geons (RCS) rats which, similarly to rd1 mice, are considered asmodels for R. pigmentosa. The two models differ in the genesinvolved: whereas rd1 mice carry a rod-specific mutation in theb subunit of cGMP-dependent phosphodiesterase causing death ofthese photoreceptors (Bowes et al., 1990), dystrophic RCS rats dis-play a mutation in the Mertk gene located in the retinal pigmentedepithelium (RPE) resulting in deficient phagocytosis of photo-receptor outer segments (La Vail et al., 1975; D’Cruz et al., 2000).Furthermore, in rd1 mice photoreceptor degeneration starts beforeeye opening whereas in dystrophic RCS rats the degenerative pro-cesses begin slowly during the third week of postnatal life witha peak between postnatal day (PNd) 40 and PNd60.

2. Materials and methods

Experiments were carried out in accordance with the EuropeanCommunities Council Directive 1986 (86/609/EEC) and the Asso-ciation for Research in Vision and Ophthalmology (ARVO) state-ment for the use of animals in ophthalmic and vision research.

A total of 80 rats (40 dystrophic and 40 non dystrophic congenicrats) were used in this study. They were sacrificed at PNd0, 7, 10, 20,30, 40, 60 and 1 year. Food and water were provided ad libitum andthe animals were maintained on a 12 h light/dark cycle. Sacrifice ofall animals was performed by carbon dioxide inhalation always at4 PM to avoid possible circadian variations in nestin expression.

2.1. Immunohistochemistry

After sacrifice, animals were enucleated and the eyes were fixedin 4% paraformaldehyde in phosphate buffer saline (PBS) (pH 7.2/7.4) for 24 h at 4 �C. Eyes were transferred to PBS containing 30%sucrose for 24 h at 4 �C then frozen in Tissue-Tek� OCT, and 12 mmsections were prepared by cryostat. Sections were mounted onclean slides coated with gelatin and washed for 20 min at roomtemperature in PBS (pH 7.2/7.4) containing 10% fetal bovine serumfor saturation of non-specific sites and 0.1% Triton X-100 (PBSST) forpermeabilization. Following incubation with an anti-nestin anti-body (1/400 antibody goat anti-r-nestin R&D-System-France)overnight at 4 �C, the sections were rinsed in PBS and covered withthe secondary antibody (rabbit anti-goat Alexa 488 e Invitrogen-France, diluted at 1/200) for 2 h at room temperature. Sectionswere washed in PBS. To identify mature Müller cells, some sectionswere prepared for double immunolabelling using anti-glutaminesynthetase (GS) (mouse anti-GS, Millipore-France) diluted at 1/

Please cite this article in press as: Valamanesh, F., et al., Nestin expressionEye Research (2013), http://dx.doi.org/10.1016/j.exer.2013.01.013

400. Immunoreactivity was detected by incubation of sections indonkey anti-mouse Alexa 594 diluted at 1/200 (Invitrogen-France).Negative controls were performed by incubating the sections in PBSinstead of the primary antibody. Sections were examined usinga photomicroscope Olympus BX51 equipped with a DP70 camera.

2.2. Western-blotting study

The time course of nestin expression during postnatal develop-ment and in adult dystrophic and control animals was assessed byWestern blotting. Animalswere killed bydecapitation and retinas (3for each age) were taken at the same age as for immunohis-tochemistry. The samples were stored at �80 �C until processing.Isolated retinas were lysed in Radio-Immunoprecipitation Assay(RIPA) buffer for 30min at 4 �C and equal amounts of the cell lysates(30e50 mg of proteins) were electrophoresed in sodium dodecylsulfatee polyacrylamide gel electrophoresis (SDS-PAGE). Separatedproteins were electrotransferred on polyvinyl difluoride (PVDF)membranes, which were further incubated with anti-r-Nestin pri-mary antibody (R&D-System-France,1/1000) or anti-bActin (Sigma,France,1/1000) for protein contentmonitoring and standardization.Following incubation with the secondary HRP antibodies (perox-idase labeled anti-goat and peroxidase labeled anti-mouse at 1/5000, Vector, France) detection was performed using enhancedchemiluminescence Detection Kit (Amersham Bioscience e LifeScience e France). The band intensities were quantified using theNIH Image 1.62b Software. Nestin expression datawas expressed asrelative to b-actin. All datawere evaluatedwith Anova and Student’st-test. Differences were considered significant at P < 0.05.

2.3. Proteasomal activity assay

Three retinas of 1-year-old dystrophic and control RCS rats(n ¼ 3 each) were used. PBS (300 ml) containing 5 mM EDTA (PBSE)was added to 1 retina and homogenized. Tissue lysates were cen-trifugated at 13,000 g for 5 min at 4 �C. The supernatants weresubjected to protein quantification employing the BCA proteinassay kit (Pierce-Thermo Scientific) with bovine serum albumin asstandard.

The supernatants were diluted to a concentration of 0.2 mg/mltotal protein with PBS. Fifty ml (corresponding to 10 mg of protein)were added to 50 ml of the luminescent reagent containing theUltra-Glo Luciferase and the signal peptide (specific for chymotrypsin-likeactivity) coupled to luciferin. After preincubation for 60min at roomtemperature, the resulting luminescence was measured three timeswith a microplate reader cytofluorometer (Wallac Victor-2 PerkineElmer, Norwalk). Luminescence signal intensity in each superna-tant is proportional to peptidase activity (i.e. the sum of the protea-somal activity and unspecific peptidase activities resulting fromother enzymes in theprotein extract). To isolate proteasomal activity,dual measurements with or without the addition of 30 mM of theirreversible and highly specific proteasomal inhibitor adamantine-acetyl-(6-aminohexanoyl) 3-vinyl-(methyl)-sulfone (AdaAhx3L3VS,Calbiochem) were carried out. The proteasomal activity was calcu-lated by subtracting the non-specific background activity (i.e. thevalue with added proteasomal inhibitor) from the total peptidaseactivity (i.e. the value without inhibitor).

2.4. Reverse transcription and quantitative polymerase chainreaction (qPCR) analysis

Three retinas for each stage were used. All products used wereprovided by Invitrogen e France. For reverse transcription, we used1 mg of total RNAs. First, total RNAs were added to a mix containing1.6 ml Random Primers (diluted at 300 mg/ml), 1 ml 10 mM dNTPs


Fig. 1. (AeC): Nestin expression in newborn (PNd0) control and dystrophic rat retina. Nestin in control (A) and dystrophic (B) RCS rat retinas. Nestin immunoreactivity (green) isfound in radial fibers spanning the retina from the inner to outer layer. GS/Nestin co-localization (C). GS immunoreactivity (red) is found in rare cell bodies and processes principallylocated in the inner retina (small horizontal arrow) Double-GS-nestin immunolabelled structures are observed in processes located in the GCL (large vertical arrow) in both control(presented here) and dystrophic animals. Abbreviations: ganglion cell layer (GCL), neuroblastic layer (NL), retinal pigment epithelium (RPE), Choroidal layer (ChL) Scale bar: 50 mm.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

F. Valamanesh et al. / Experimental Eye Research xxx (2013) 1e9 3

241242243244245246247248249250251252253254255256257258259260261262263264265266267268269270271272273274275276277278279280281282283284285286287288289290291292293294295296297298299300301302303304305

306307308309310311312313314315316317318319320321322323324325326327328329330331332333334335336337338339340341342343344345


and completed to 12 ml by RNAse-free distilled water. After incu-bation for 5 min at 65 �C, 4 ml of 5X First Buffer (Invitrogen-France)and 1 ml RNase OUT (Recombinant Ribonuclease Inhibitors 40 units/ml) were added. The mix was incubated for 2 min at 45 �C. Finally,1 ml Superscript II reverse transcriptase was added. After incubationfor 50min at 42 �C, then for 15min at 70 �C, 200 ml RNase freewaterwas added. Products were analyzed using the following TaqMan�

gene expression assays (Applied Biosystems): 18S, Hs99999901_s1as endogenous control and nestin, Rn00564394_m1 for rat(Applied Biosystems, France). Detectionwas performed with 5 ml ofsample in 20 ml of mix containing 12.5 ml Taqman, 1.25 ml primers(nestin or 18S) and 6.25 ml RNase free distilled water. Cycling pa-rameters for qPCR were as follows: initial denaturation 95 �C for10 min, followed by 40 cycles of 15 s at 95 �C (denaturation), 1 minat 60 �C (hybridization) and 2 min at 72 �C (elongation). QPCRs

Fig. 2. (AeC): Nestin expression in PNd7 control and dystrophic rat retina. Nestin in controlocated in elongated radial fibers but immunolabelling is more intense in the inner than in oblood vessels. GS/Nestin co-localization (C). GS immunoreactivity (red) is found in rare cell bocells present at this stage. Co-localization of nestin and GS immunolabellings is seen in pdystrophic animals. Abbreviations: ganglion cell layer (GCL), inner plexiform layer (IPL), inne50 mm. (For interpretation of the references to color in this figure legend, the reader is refe


were performed using a Real Time PCR System 7300 (AppliedBiosystem). The PCR efficiency of the reactionwas measured by theDD Ct method.

3. Results

3.1. Expression of nestin during postnatal rat retinal development

At PNd0, neural retinas of rodents are composed of two majorlayers: a large outer layer with neuroblastic cells and an inner layercontaining differentiated ganglion cells. No morphological differ-ences were observed between the retinas of control and dystrophicrats. Nestin immunoreactivity was localized in numerous elongatedradial processes spanning the thickness of the retina (Fig. 1A, B).The high level of nestin immunoreactivity was confirmed by

l (A) and dystrophic (B) RCS rat retinas. As in PNd0, nestin immunoreactivity (green) isuter layers (star). Note that immunolabelling is also observed in the ChL which containsdies located in INL (small horizontal arrow). These cells are probably rare mature Müllerrocesses within the GCL (large vertical arrow), in both control (presented here) andr nuclear layer (INL), retinal pigment epithelium (RPE) choroidal layer (ChL). Scale bar:rred to the web version of this article.)


346347348349350351352353354355356357358359360361362363364365366367368369370

Fig. 3. (AeC): Nestin expression in PNd10 control and dystrophic rat retinas. Nestin incontrol (A) and dystrophic (B) RCS rat retinas. As in previous stages, nestin immu-noreactivity (green) is located in elongated radial fibers and immunolabelling is moreintense in inner than in outer layers (star). Nestin immunoreactivity can be alsoobserved into the ChL which contains many blood vessels (arrow head). GS/nestin co-localization (C1, C2). The pattern of nestin immunolabelling is similar in control(presented here) and in dystrophic rat retina. As shown in Fig. C1, GS immunoreactivity(red) is clearly observed in Muller cells (small vertical arrow) and GS/nestin co-localization is only seen in processes located in the GCL (large arrows). Fig. C2 isa magnified view of the GCL. Abbreviations: ganglion cell layer (GCL), inner plexiformlayer (IPL), inner nuclear layer (INL), OPL (outer plexiform layer), ONL (outer nuclearlayer), choroidal layer (ChL). Scale bar: 50 mm for A, B and C1 and 10 mm for C2. (Forinterpretation of the references to color in this figure legend, the reader is referred tothe web version of this article.)


371372373374375376377378379380381382383384385386387388389390391392393394395396397398399400401402403404405406407408409410411412413414415416417418419420421422423424425426427428429430431432433434435

436437438439440441442443444445446447448449450451452453454455456457458459460461462463464465466467468469470471472473474475476477478479480481482483484485486487488489490491492493494495496497498499500


Western-blotting (Fig. 7A, B) and qPCR analysis (Fig. 8A, B). GSimmunoreactive processes and cell bodies probably belonging toastrocytes were identified in the ganglion cell layer (GCL). Rare GS/nestin co-immunolabelled cells were observed in the GCL (Fig. 1C).

At PNd7, nestin immunoreactivity was again principally detec-ted in radial processes. However, although the intensity of immu-nostaining remained strong in the inner retina, it was reduced inthe emerging outer nuclear layer (ONL) (Fig. 2A, B). Nestin immu-noreactivity was correlated with Western blotting and qPCR re-sults: although levels remained high, slight reductions in nestinprotein andmRNA contents were observed in dystrophic comparedto control animals (Figs. 7A, B and 8A, B). Rare GS positive cells withthe morphology of Müller cells were detected. The number of GS/nestin co-immunolabelled structures was increased compared toPNd0 but remained restricted to processes located in the GCL inboth strains (Fig. 2C).

At PNd10, all retinal layers were distinguishable in both controland dystrophic RCS rats. Nestin immunolabelling was localized


principally in the inner part of radial processes, as previouslyobserved at PNd7. Choroidal blood vessels were also partiallylabeled (Fig. 3A, B). Nestin content decreased in both dystrophicand control rats as shown by Western blotting (Fig. 7A, B) andconfirmed by qPCR (Fig. 8A, B). Mature Müller cells stained by theanti-GS antibody were now clearly identifiable. GS/nestin co-immunolabelling was only observed in processes located in theGCL in both strains (Fig. 3C).

3.2. Expression of nestin during retinal maturation and adulthoodin control and dystrophic rats

At PNd20, the retina of dystrophic rats presented a normalmorphological appearance and the number of radial nestin positiveprocesses was considerably decreased. Immunostaining was foundin rare processes located in the GCL in both control and dystrophicanimals. Some retinal blood vessels were also partially labeled bynestin, in both retina and choroid (Fig. 4AeC). The decrease innestin content was confirmed by the Western blotting (Fig. 7A, B)and qPCR analysis (Fig. 8A, B). GS-positive mature Müller cells andastrocytes were clearly identifiable and GS/nestin co-immunolabelling was still found principally in a subpopulation ofprocesses located in the GCL (Fig. 4C).

Distribution and intensity of nestin immunostaining were sim-ilar at PNd30 (Results no shown), corroborated byWestern blotting(Fig. 7A, B) and qPCR analysis (Fig. 8A, B).

Whereas no change was observed in retinal morphology anddistribution of nestin immunoreactivity in control animals, pho-toreceptor degeneration was apparent in dystrophic rats at PNd40(Fig. 5AeD) and PNd60 (not shown). A debris zone (DZ), containingdegenerating and/or microglial cells surrounded the outer face ofthe retina (Fig. 5B, D). Numerous radial nestin-immunoreactiveprocesses spanning the retina were identified. The presence ofnestin immunolabelling surrounding some blood vessels was alsodetected in both control and dystrophic rats (Fig. 5A, B). Westernblot and qPCR studies confirmed that protein (Fig. 7A, B) andmRNAcontents (Fig. 8A, B) were low in control rats and higher in dys-trophic rats. GS immunoreactivity was found in Müller and astro-glial cells of all rats. In both control and dystrophic retinas, GS-nestin co-staining was observed principally in the GCL, in innerradial processes. However, rare radial glial cell bodies exhibiteddouble GS/nestin immunolabellings. Some showed alternating GSand nestin immunolabelling along their lengths (Fig. 5D1, D2).

At 1 year (Fig. 6AeC) nestin immunoreactivity was very sparselydistributed around some blood vessels and in processes locatedwithin the inner retina of control animals (Fig. 6A). In dystrophicRCS rats, the photoreceptor layer was entirely degenerated to bereplaced by the DZ. Nestin was present in numerous radial pro-cesses spanning the thickness of the remnant retina. The DZ layeralso contained nestin-immunostained structures (probably micro-glial cells and degenerating cells) (Fig. 6B). Whereas the levels ofnestin protein and mRNAs remained low in control rats a discrep-ancy appeared in dystrophic rat: the protein content was high andthe level of mRNAs remained low, comparable to that found incontrol rats (Figs. 7A, B and 8A, B). GS-immunoreactivity wasobserved in numerous Müller cells in both dystrophic and controlrat retina. As in previous stages, GS/nestin co-immunolabelling wasfound principally in processes located in the inner retina, and as atPNd40 some radial processes exhibited alternating immunor-eactivity to nestin and GS (Fig. 6C).

To attempt to explain the discrepancy between the high proteinlevel compared to the low level of nestin mRNA, we measured theproteasomal activity by a luminescence-based technique. We noteda significant decrease in the specific proteasomal activity in dys-trophic 1 year-old rats compared to control rats (Fig. 9).


Fig. 4. (AeC): Nestin expression in PNd20 control and dystrophic RCS rat retina. Nestin in control (A) and dystrophic (B) RCS rat retinas. Nestin immunoreactivity (green) isobserved only in processes located in the GCL in both rat strains (small oblique arrow). Nestin immunolabelling is present in some blood vessels (arrow head). GS/nestin co-localization (C). GS immunoreactivity (red) is seen in numerous Muller cells (small horizontal arrow). GS/nestin co-immunolabelling is observed in some processes located in theGGL in both control and dystrophic (presented here) rat retina (large vertical arrow). Note that nestin immunoreactivity is also found in blood vessels located in the ChL (arrow head)in both dystrophic (presented here) and control rat. Abbreviations: ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), OPL (outer plexiform layer), ONL(outer nuclear layer), choroidal layer (ChL). Scale bar: 50 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of thisarticle.)


501502503504505506507508509510511512513514515516517518519520521522523524525526527528529530531532533534535536537538539540541542543544545546547548549550551552553554555556557558559560561562563564565

566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607608609610611612613614615616617618619620621622623624625626627628629630


4. Discussion

Nestin is a large intermediate filament protein (>1600 aminoacids), originally described as a neuronal stem cell/progenitor cellmarker during CNS development (Lendahl et al., 1990). It is nowknown to be expressed also in progenitor cells in several normaland pathological adult tissues (Sejersen and Lendahl, 1993;Frojdman et al., 1997; Hoffman, 2007; Yamada et al., 2009), inwhich it is widely used as a marker for adult stem cells thatcharacteristically display features such as multipotency, self-renewal and regeneration. However, although some studiesreport its involvement in cytoskeletal organization, coordinatedchanges in cell dynamics and modulation of mitosis-associatedreorganization (Herrmann and Aebi, 2000), its function(s)remain incompletely understood. In this study, we show thatnestin is transiently expressed in radial glia during postnataldevelopment in both dystrophic and control RCS rats, and that it isre-expressed in cells with similar morphology only in pathologicaladult retinas.

4.1. Transient expression of nestin during the first week of thepostnatal retinogenesis

Nestin is observed as soon as birth in radial processes in theretina of both dystrophic and normal rats. These processes maybelong to radial glia because during neural tube formation nestinis found in radial processes which guide neurons to their appro-priate layer (Park et al., 2009). However, it has been proposed thatsome of these processes could be Müller cells. Indeed some au-thors suggest that these cells are already present at embryonicstages (Bhattacharjee and Sanyal, 1975) and/or progressivelyemerge during the first week after birth (Kimura et al., 2000). Weconfirm the presence of functionally mature Müller cells (asattested by the presence of GS) only by the end of the first post-natal week in control and dystrophic RCS rats, as previouslydemonstrated (Riepe and Norenberg, 1978; Xue et al., 2006b).Before this stage, GS immunolabelling is seen in rare isolatedperikarya and in short processes probably belonging to astrocytes,as suggested by Riepe and Norenberg (1978). The lack of GSimmunoreactivity in radial processes during the first week


confirms that, if Müller cells are present in the retina, they are inthe progenitor state and functionally immature. Consequently, inearly retinal development Müller cell progenitors and retinal radialglia may be overlapping populations (Walcott and Provis, 2003;Xue et al., 2006b).

During the first week of postnatal life, the morphological profileof retinal development appears globally identical in both dystro-phic and control RCS rat. However, some slight physiological dif-ferencesmay exist as suggested by the variations observed in nestinprotein and mRNA contents at PNd7. The relatively high levels ofprotein and mRNAs in control compared to dystrophic RCS ratscould be explained by the pre-existing pathology. It could also bedue to the degree of pigmentation in our study, control rats werealbino and dystrophic rats were pigmented, and it is known thatthe retina of albino rats develops a little later than pigmented rats(Webster and Rowe, 1991).

4.2. Transient expression of nestin from the second week of thepostnatal retinogenesis

As previously demonstrated by Wu et al. (2004), retinal nestinexpression is progressively down regulated from the beginning ofthe second postnatal week. Initially observed in the outer retina,the disappearance of radial nestin-immunolabelled processescontinues in the inner retina to the end of the third week of post-natal life, in accordance with the progressive morphological andfunctional maturation of retina in both strains. Nestin down-regulation is similarly observed in the brain after cellular differ-entiation (Voigt, 1989; Wei et al., 2002). At PNd20, only somenestin-containing processes are observed in the GCL. These pro-cesses may belong to either Müller cell end-feet or astrocytes asindicated by GS/nestin co-immunolabellings.

4.3. Nestin re-expression in adult rodent retina with inheritedprogressive retinal degeneration

The obvious difference between normal and pathological ani-mals consists in the expression of nestin in dystrophic rat retinaswhen the degeneration of photoreceptors begins (from PNd30/PN40). These results confirm previous multiple studies which


Fig. 5. (AeD): Nestin expression in PNd40 control and dystrophic rat retina. Nestinin control (A) and dystrophic (B) RCS rat retina. As in the previous stage, nestinimmunoreactivity (green) is present in processes located in GCL (small obliquearrow) and in some blood vessels (arrow head) in the control rat retina. In thedystrophic retina, the photoreceptor layer is undergoing degeneration. Nestinimmunoreactivity is found in numerous radial processes spanning the retina. Someimmunolabelled structures (probably microglial cells and cellular debris) areobserved in DZ of dystrophic rats. GS/Nestin co-localization in control (C) anddystrophic (D1-D2) rat. GS/nestin co-localization is found principally in the GCL(large arrow). Few radial processes are entirely co-immunolabelled. Most of theseprocesses are exclusively nestin-immunopositive, or are partially and alternativelylabeled by one and other of the antibodies (Fig. D1). The Fig. D2 highlights thisalternating immunolabelling (double arrows). Abbreviations: debris zone (DZ),ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), OPL(outer plexiform layer), ONL (outer nuclear layer). Scale bar: 50 mm. (For inter-pretation of the references to color in this figure legend, the reader is referred to theweb version of this article.)


631632633634635636637638639640641642643644645646647648649650651652653654655656657658659660661662663664665666667668669670671672673674675676677678679680681682683684685686687688689690691692693694695

696697698699700701702703704705706707708709710711712713714715716717718719720721722723724725726727728729730731732733734735736737738739740741742743744745746747748749750751752753754755756757758759760


report an increase in the nestin content after an acute or inheriteddegenerative pathology (Fischer and Omar, 2005; Xue et al., 2006a,2006b; Chang et al., 2007; Karl et al., 2008; Wan et al., 2008; Goeland Dhingra, 2012). It has been suggested that the over-expressionof nestin could be due tometabolic changes in neighboring neuronsin response to the degeneration. Little is known about the factorscontrolling nestin induction but b-fibroblast growth factor (bFGF),insulin growth factor (IGF), ciliary neurotrophic growth factor(CNGF), basic fibroblast growth factor (bFGF) seem to be involved(Lewis et al., 1992; Cao et al., 2001; Fischer et al., 2004). A reac-tivation of such factors must be considered in the retina of dys-trophic RCS rats.


4.4. Possible nature and functions of cells re-expressing nestin

Nestin, a marker for proliferative and progenitor cells is princi-pally re-expressed in radial cells resembling Müller cells. However,because of the scarcity of GS/nestin co-immunolabelled cells, it canbe hypothesized that the cells which express nestin are de-differentiated Müller cells. Although questioned by some authors(Tackenberg et al., 2009), Müller cell de-differentiation is suggestedto constitute a step in regeneration of adult retinal neurons. Inchickens and rodents, Müller cells have been shown to re-enter thecell cycle prior to re-differentiation into retinal neurons followinginjury or in response to intraocular injections of growth factors(Fischer and Reh, 2001, 2002; Ooto et al., 2004; Karl et al., 2008;Wan et al., 2008). Recent studies have demonstrated that a sub-population of Müller cells tends to re-differentiate selectively intorods after these cells degenerate during an inherited retinal pa-thology (Goel and Dhingra, 2012). Although the functionality andintegration of these cells remains open to question, Müller cellshave neural stem cell properties as confirmed by in vitro experi-ments demonstrating the production of neurospheres from Müllercells (Das et al., 2006; Kubota et al., 2006; Monnin et al., 2007).These neurospheres are self-renewing and multipotent cells thatare capable of generating neurons in vitro and site-specific neuronsupon transplantation (Das et al., 2006).

Because of its cytoskeletal nature, nestin would also providea stronger rigidity of Müller glial cells to strengthen the retinalarchitecture that would be disrupted by neuronal loss. Xue et al.(2006b) and Frisén et al. (1995) suggest that induction and up-regulation of nestin expression after retinal damage are involvedin the generation of a glial scar. Other studies suggest that radialglia could serve as guides for new neurons to migrate upon duringretinal remodeling. In this respect the re-expression of nestin mayalso indicate that Müller cells are able to de-differentiate into radialglia cells.

Nestin could also participate in atypic reactive gliosis charac-terized by the proliferation of Müller cells, as observed in rdmutantmice by Iandiev et al. (2006) and in rats after genetic and exper-imental photoreceptor degeneration (Eisenfeld et al., 1984). Gliosishas numerous functions: at early stages after injury, it is neuro-protective from further damage by the release of neurotrophic fac-tors and antioxidants. At later stages, it contributes to neuronal celldeath via impairment of neurotransmitter removal and dysregula-tion of the ion andwater homeostasis (Bringmann andReichenbach,2001; Lewis and Fisher, 2003; Bringmann et al., 2006).

Coherent variations in protein/mRNA levels is seen in dystrophicanimals during the early stage of the disease, but when photore-ceptors are totally degenerated a discrepancy between the level ofmRNAs (low) and protein (high) content is observed. Several hy-potheses can be considered to explain such results: an increase inprotein half-life due to dysfunction in stabilization components,a disruption in protein turnover and the presence of a high level ofprotein in the DZ before degradation, stabilization throughproteineprotein interactions or a dysfunction in transcriptionprocesses and/or in proteasome action (Mellodew et al., 2004; Jinet al., 2009). Our results favor this last hypothesis since the pro-teasomal activity is significantly reduced in one year old dystrophicrats. Such proteasomal dysfunction, which induces an abnormalaccumulation of proteins, is also observed in various neuro-degenerative diseases and several retinal dystrophies (Surguchevaet al., 2005; Dohm et al., 2008).

4.5. Nestin immunoreactivity in blood vessels

We observed sporadic nestin immunolabelling along the wallsof some retinal and choroidal blood vessels in normal and


Fig. 6. (AeC): Nestin expression in one year control and dystrophic rat retina. Nestin in control (A) and dystrophic (B) RCS rat retinas. In control retina nestin (green) is present inrare processes located in GCL (small oblique arrow) and in some blood vessels (arrow head). In dystrophic retina, nestin is expressed in radial processes (small oblique arrow) andunidentified structures (as in previous stades, probably microglial cells and cellular debris) in DZ (star). GS/nestin co-localization in dystrophic (C) rats. As for precedent stages, GSimmunoreactivity (red) identifies the Muller cells (small vertical arrow). GS/Nestin co-immunolabelling is found principally in the GCL (large arrow). Radial processes spanning theretina are immunolabelled either by the nestin antibody (small oblique arrow), by the GS antibody (small vertical arrow), or as in previous stades, alternatively by nestin and GSantibodies (double arrows). Abbreviations: ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), OPL (outer plexiform layer), ONL (outer nuclear layer) andzone of debris (ZD). Scale bar: 50 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7. Western blotting analysis showing relative content (nestin/actin protein) in theretinas of control (A) and dystrophic (B) RCS rats fromPNd0 to 1 year. Note that in controlRCS rats, the relative retinal content of nestin decreases from PNd 7 to PNd20, and re-mains low to 1 year. In dystrophic RCS rats, the relative nestin content decreases stronglyfrom PN0 to PN10, and thenmore gradually between PNd10 and PNd30. Photoreceptorsdegeneration begins around PNd30 and the relative nestin content increases progres-sively to 1 year. Each value is compared with previous stage (P < 0.001).



761762763764765766767768769770771772773774775776777778779780781782783784785786787788789790791792793794795796797798799800801802803804805806807808809810811812813814815816817818819820821822823824825

826827828829830831832833834835836837838839840841842843844845846847848849850851852853854


dystrophic animals from PNd20. Although such staining has beenthought to be artefactual by some authors (Luna et al., 2010), nestinis now considered as a marker of proliferative endothelial cells(Teranishi et al., 2007; Suzuki et al., 2010). Its presence may be

Fig. 8. Q-PCR analysis showing relative content (nestin/18S) in the retinas of control(A) and dystrophic (B) RCS rats from PNd0 to 1 year. In control RCS rats, the relativecontent of nestin mRNAs is high at PNd0 and PNd 7 decreasing to PNd20. Valuesremain stably low to PNd60, and are considerably reduced at 1 year. In dystrophic RCSrats, the relative decrease in nestin mRNAs content is progressive from PNd0, toPNd20. After this stage, the levels increase slightly from PNd40 and 60, thendecreasing to reach a value near zero to 1 year. The mRNAs concentration profiles arevery similar to protein contents in all animals, except at 1 year dystrophic retinas(***P < 0.05). For each stage, the statistical significance of the results is calculatedrelatively to PNd0.


855856857858859860861862863864865866867868869870871872873874875876877878879880881882883884885886887888889890

Fig. 9. Proteasome activity in control and dystrophic rats at 1 year demonstrated byrelative units of luminescence. Note the significative decrease (P < 0.05) in the pro-teasome activity in dystophic compared to control rat retina.


891892893894895896897898899900901902903904905906907908909910911912913914915916917918919920921922923924925926927928929930931932933934935936937938939940941942943944945946947948949950951952953954955

956957958959960961962963964965966967968969970971972973974975976977978979980981982983984985986987988989990991992993994995996997998999

100010011002100310041005100610071008100910101011101210131014101510161017101810191020


correlated with the degree of normal and permanent proliferationof the endothelial cells throughout the life of retina.

In conclusion, we found no major differences in nestin expres-sion in probable radial glia during the first 3 weeks of postnatalretinal development in both control and dystrophic RCS rats. Inadulthood, while nestin levels are low in control animals, weobserved a re-expression of nestin protein resembling radial glia orimmature Müller cells in dystrophic animals. We show that theincrease in nestin protein content observed in late stage pathologymay be correlated with defective protein degradation oftenobserved in neurodegenerative disease.

Acknowledgments

This studywas supported, in part, by a grant of “Conseil Régionalde Franche-Comté”. The authors are grateful to Prof. Patrick Ver-guet and Dr. David Hicks for insightful comments.

References

Ahmad, I., Tang, L., Pham, H., 2000. Identification of neural progenitors in the adultmammalian eye. Biochem. Biophys. Res. Commun. 270, 517e521.

Bhattacharjee, J., Sanyal, S., 1975. Developmental origin and early differentiation ofretinal Müller cells in mice. J. Anat. 120, 367e372.

Bowes, C., Li, T., Danciger, M., Baxter, L.C., Applebury, M.L., Farber, D.B., 1990. Retinaldegeneration in the rd mouse is caused by a defect in the beta subunit of rodcGMP-phosphodiesterase. Nature 347, 677e680.

Bringmann, A., Reichenbach, A., 2001. Role of Müller cells in retinal degenerations.Front. Biosci. 6, E72eE92 (Review).

Bringmann, A., Pannicke, T., Grosche, J., Francke, M., Wiedemann, P., Skatchkov, S.N.,Osborne, N.N., Reichenbach, A., 2006. Müller cells in the healthy and diseasedretina. Prog. Retin. Eye Res. 25, 397e424 (Review).

Cao, W., Li, F., Steinberg, R.H., Lavail, M.M., 2001. Development of normal and injury-induced gene expression of aFGF, bFGF, CNTF, BDNF, GFAP and IGF-I in the ratretina. Exp. Eye Res. 72, 591e604.

Chang, M.L., Wu, C.H., Jiang-Shieh, Y.F., Shieh, J.Y., Wen, C.Y., 2007. Reactive changesof retinal astrocytes and Müller glial cells in kainate-induced neuro-excitotoxicity. J. Anat. 210, 54e65.

Das, A.V., Mallya, K.B., Zhao, X., Ahmad, F., Bhattacharya, S., Thoreson, W.B.,Hegde, G.V., Ahmad, I., 2006. Neural stem cell properties of Müller glia in themammalian retina: regulation by Notch andWnt signaling. Dev. Biol. 299, 283e302.

D’Cruz, P.M., Yasumura, D., Weir, J., Matthes, M.T., Abderrahim, H., LaVail, M.M.,Vollrath, D., 2000. Mutation of the receptor tyrosine kinase gene Mertk in theretinal dystrophic RCS rat. Hum. Mol. Genet. 9, 645e651.

Dohm, C.P., Kermer, P., Bähr, M., 2008. Aggregopathy in neurodegenerative diseases:mechanisms and therapeutic implication. Neurodegener. Dis. 5, 321e338.

Eisenfeld, A.J., Bunt-Milam, A.H., Sarthy, P.V., 1984. Müller cell expression of glialfibrillary acidic protein after genetic and experimental photoreceptor degen-eration in the rat retina. Invest. Ophthalmol. Vis. Sci. 25, 1321e1328.

Fischer, A.J., Reh, T.A., 2001. Müller glia is a potential source of neural regenerationin the postnatal chicken retina. Nat. Neurosci. 4, 247e252.

Fischer, A.J., Reh, T.A., 2002. Exogenous growth factors stimulate the regeneration ofganglion cells in the chicken retina. Dev. Biol. 25, 367e379.

Fischer, A.J., Omar, G., Eubanks, J., McGuire, C.R., Dierks, B.D., Reh, T.A., 2004. Dif-ferent aspects of gliosis in retinal Müller glia can be induced by CNTF, insulin,and FGF2 in the absence of damage. Mol. Vis. 10, 973e986.


Fischer, A.J., Omar, G., 2005. Transitin, a nestin-related intermediate filament isexpressed by neural progenitors and can be induced in Müller glia in thechicken retina. J. Comp. Neurol. 484, 1e14.

Frisén, J., Johansson, C.B., Török, C., Risling, M., Lendahl, U., 1995. Rapid, widespread,and longlasting induction of nestin contributes to the generation of glial scartissue after CNS injury. J. Cell. Biol. 131, 453e464.

Frojdman, K., Pelliniemi, L.J., Lendahl, U., Virtanen, I., Eriksson, J.E., 1997. The in-termediate filament protein nestin occurs transiently in differentiating testis ofrat and mouse. Differentiation 61, 243e249.

Goel, M., Dhingra, N.K., 2012. Müller glia expresses rhodopsin in a mouse model ofinherited retinal degeneration. Neuroscience 225, 152e161.

Herrmann, H., Aebi, U., 2000. Intermediate filaments and their associates: multi-talented structural elements specifying cytoarchitecture and cytodynamics.Curr. Opin. Cell. Biol. 12, 79e90.

Hockfield, S., McKay, R.D., 1985. Identification of major cell classes in the developingmammalian nervous system. J. Neurosci. 5, 3310e3328.

Hoffman, R.M., 2007. The potential of nestin-expressing hair follicle stem cells inregenerative medicine. Expert Opin. Biol. Ther. 7, 289e291.

Iandiev, I., Biedermann, B., Bringmann, A., Reichel, M.B., Reichenbach, A.,Pannicke, T., 2006. Atypical gliosis in Müller cells of the slowly degenerating rdsmutant mouse retina. Exp. Eye Res. 82, 449e457.

Jin, Z., Liu, L., Bian, W., Chen, Y., Xu, G., Cheng, L., Jing, N., 2009. Different tran-scription factors regulate nestin gene expression during P19 cell neural differ-entiation and central nervous system development. J. Biol. Chem. 284, 8160e8173.

Kachinsky, A.M., Dominov, J.A., Miller, J.B., 1995. Intermediate filaments in cardiacmyogenesis: nestin in the developing mouse heart. J. Histochem. Cytochem. 43,843e847.

Karl, M.O., Hayes, S., Nelson, B.R., Tan, K., Buckingham, B., Reh, T.A., 2008. Stimu-lation of neural regeneration in the mouse retina. Proc. Natl. Acad. Sci. U S A105, 19508e19513.

Kimura, N., Nishikawa, S., Tamai, M., 2000. Müller cells in developing rats withinherited retinal dystrophy. Tohoku J. Exp. Med. 191, 157e166.

Kohno, H., Sakai, T., Kitahara, K., 2006. Induction of nestin, Ki-67, and cyclin D1expression in Müller cells after laser injury in adult rat retina. Graefes Arch.Clin. Exp. Ophthalmol. 244, 90e95.

Kubota, A., Nishida, K., Nakashima, K., Tano, Y., 2006. Conversion of mammalianMüller glial cells into a neuronal lineage by in vitro aggregate-culture. Biochem.Biophys. Res. Commun. 351, 514e520.

La Vail, M.M., Sidman, R.L., Gerdhardt, C.O., 1975. Congenic strains of RCS rats withinheritedretinal dystrophy. J. Hered. 66, 242e244.

Lendahl, U., Zimmerman, L.B., McKay, R.D., 1990. CNS stem cells express a new classof intermediate filament protein. Cell 60, 585e595.

Lewis, G.P., Erickson, P.A., Guérin, C.J., Anderson, D.H., Fisher, S.K., 1992. Basicfibroblast growth factor: a potential regulator of proliferation and intermediatefilament expression in the retina. J. Neurosci. 12, 3968e3978.

Lewis, G.P., Fisher, S.K., 2003. Up-regulation of glial fibrillary acidic protein inresponse to retinal injury: its potential role in glial remodeling and a compar-ison to vimentin expression. Int. Rev. Cytol. 230, 263e290 (Review).

Luna, G., Lewis, G.P., Banna, C.D., Skalli, O., Fisher, S.K., 2010. Expression profiles ofnestin and synemin in reactive astrocytes and Müller cells following retinalinjury: a comparison with glial fibrillar acidic protein and vimentin. Mol. Vis. 16,2511e2523.

Mellodew, K., Suhr, R., Uwanogho, D.A., Reuter, I., Lendahl, U., Hodges, H., Price, J.,2004. Nestin expression is lost in a neural stem cell line through a mechanisminvolving the proteasome and notch signalling. Brain Res. Dev. Brain Res. 15,13e23.

Monnin, J., Morand-Villeneuve, N., Michel, G., Hicks, D., Versaux-Botteri, C., 2007.Production of neurospheres from mammalian Müller cells in culture. Neurosci.Lett. 421, 22e26.

Ooto, S., Akagi, T., Kageyama, R., Akita, J., Mandai, M., Honda, Y., Takahashi, M., 2004.Potential for neural regeneration after neurotoxic injury in the adult mamma-lian retina. Proc. Natl. Acad. Sci. U S A 101, 13654e13659.

Park, D., Xiang, A.P., Zhang, L., Mao, F.F., Walton, N.M., Choi, S.S., Lahn, B.T., 2009. Theradial glia antibody RC2 recognizes a protein encoded by nestin. Biochem.Biophys. Res. Commun. 382, 588e592.

Riepe, R.E., Norenberg, M.D., 1978. Glutamine synthetase in the developing ratretina: an immunohistochemical study. Exp. Eye Res. 27, 435e444.

Sejersen, T., Lendahl, U., 1993. Transient expression of the intermediate filamentnestin during skeletal muscle development. J. Cell. Sci. 106, 1291e1300.

Surgucheva, I., Ninkina, N., Buchman, V.L., Grasing, K., Surguchov, A., 2005. Proteinaggregation in retinal cells and approaches to cell protection. Cell. Mol. Neu-robiol. 25, 1051e1066.

Suzuki, S., Namiki, J., Shibta, S., Mastuzaki, Y., Okano, H., 2010. The neural stem/progenitor cell marker nestin is expressed in proliferative endothelial cells butnot in mature vasculature. J. Histochem. Cytochem. 58, 721e730.

Tackenberg, M.A., Tucker, B.A., Swift, J.S., Jiang, C., Redenti, S., Greenberg, K.P.,Flannery, J.G., Reichenbach, A., Young, M.J., 2009. Müller cell activation, prolif-eration and migration following laser injury. Mol. Vis. 15, 1886e1896.

Teranishi, N., Naito, Z., Ishiwata, T., Tanaka, N., Furukawa, K., Seya, T., Shinji, S.,Tajiri, T., 2007. Identification of neovasculature using nestin in colorectal cancer.Int. J. Oncol. 30, 593e603.

Terling, C., Rass, A., Mitsiadis, T.A., Fried, K., Lendahl, U., Wroblewski, J., 1995.Expression of the intermediate filament nestin during rodent tooth develop-ment. Int. J. Dev. Biol. 39, 947e956.



102110221023102410251026102710281029103010311032

103310341035103610371038103910401041104210431044


Tropepe, V., Coles, B.L., Chiasson, B.J., Horsford, D.J., Elia, A.J., McInnes, R.R., van derKooy, D., 2000. Retinal stem cells in the adult mammalian eye. Science 287,2032e2036.

Voigt, T.,1989. Developmentof glial cells in the cerebralwall of ferrets: direct tracingoftheir transformation from radial glia into astrocytes. J. Comp. Neurol. 289, 74e88.

Walcott, J.C., Provis, J.M., 2003. Müller cells express the neuronal progenitor cellmarker nestin in both differentiated and undifferentiated human foetal retinas.Clin. Exp. Ophthalmol. 31, 246e249.

Wan, J., Zheng, H., Chen, Z.L., Xiao, H.L., Shen, Z.J., Zhou, G.M., 2008. Preferentialregeneration of photoreceptor from Müller glia after retinal degeneration inadult rat. Vis. Res. 48, 223e234.

Webster, M.J., Rowe, M.H., 1991. Disruption of developmental timing in the albinorat retina. J. Comp. Neurol. 307, 460e474.

Wei, L.C., Shi, M., Chen, L.W., Cao, R., Zhang, P., Chan, Y.S., 2002. Nestin-containingcells express glial fibrillary acidic protein in the proliferative regions of central


nervous system of postnatal developing and adult mice. Brain Res. Dev. BrainRes. 139, 9e17.

Wu, J.H., Yi, M.Y., Huang, Q., 2004. The structural and temporal characteristics offour intermediate filament proteins during retinal development of rat. Zhong-hua Yan Ke Za Zhi 40, 765e769.

Xue, L.P., Lu, J., Cao, Q., Hu, S., Ding, P., Ling, E.A., 2006a. Müller glial cells expressnestin coupled with glial fibrillary acidic protein in experimentally inducedglaucoma in the rat retina. Neuroscience 139, 723e732.

Xue, L.P., Lu, J., Cao, Q., Kaur, C., Ling, E.A., 2006b. Nestin expression in Müller glialcells in postnatal rat retina and its upregulation following optic nerve trans-ection. Neuroscience 143, 117e127.

Yamada, H., Takano, T., Ito, Y., Matsuzuka, F., Miya, A., Kobayashi, K.,Yoshida, H., Watanabe, M., Iwatani, Y., Miyauchi, A., 2009. Expression ofnestin mRNA is a differentiation marker in thyroid tumors. Cancer Lett.280, 61e64.


nestin expression in the retina of rats with inherited retinal degeneration

Documents