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. 2011 Dec;92(6):377–381. doi: 10.1111/j.1365-2613.2011.00786.x

Nestin is induced by hypoxia and is attenuated by hyperoxia in Müller glial cells in the adult rat retina

Liping Xue *, Peng Ding †, Libo Xiao *, Min Hu *, Zhulin Hu *
PMCID: PMC3248073  PMID: 22050385

Abstract

This study investigated the reactive changes in Müller glial cells and astrocytes of the rat retinae, which had been subjected either to hypoxia or to hypoxia followed by hyperoxia treatments. Fifteen rats were used. Ten rats were exposed to 9% O2 for 2 h. Of these, five rats were killed at 24 h later; the remaining five rats were immediately exposed to 80% O2 for 2 h and then killed 24 h later. Double immunofluorescence was carried out between nestin and glutamine synthetase (GS) and between glial fibrilary acidic proteins (GFAP) and GS in normal and pathological retinae. Enhanced nestin expression was observed in reactive astrocytes following hypoxia treatment as revealed in whole mount sections. A novel finding was the induction of nestin expression in Müller glial cells. Remarkably, the nestin immunostaining was downregulated to levels comparable to those of the normal rats with immediate hyperoxia treatment. Induced nestin expression by hypoxia colabelled with GFAP in astrocytes, however, remained unaffected after hyperoxia treatment. The induced expression of nestin in Müller glial cells and astrocytes in hypoxia and differential downregulation after hyperoxia treatment suggest a structural plasticity of the cytoskeletal framework of these cells. The differential response after hyperoxia treatment may be related to the functional states of the cells.

Keywords: astrocytes, hyperoxia, hypoxia, Müller glial cells, nestin

The nestin gene, encoding a sixth class of intermediate filaments, was originally cloned from an E15 rat central nervous system (CNS) cDNA library (Lendahl et al. 1990). Nestin is expressed characteristically in neural stem cells (Lendahl et al. 1990; Kawaguchi et al. 2001). When neural stem cells differentiate into neuronal and glial cells, nestin is downregulated and replaced by cell type–specific intermediate filaments, i.e. neurofilaments in neurons and glial fibrilary acidic proteins (GFAP) in glia, respectively (Frederiksen & McKay 1988). However, more and more studies have shown that nestin is also expressed in other cell types, especially in reactive astrocytes when the CNS is under trauma or stress (Frisen et al. 1995; Brook et al. 1999; Clarke et al. 1999). With regard to the retina, some recent studies have shown that nestin is not only expressed in developing Müller glial cells (Fischer & Oma 2005), but it can also be induced in retinal Müller glial cells following either toxic injury (Ooto et al. 2004; Fischer & Oma 2005) or laser injury (Kohno et al. 2006). In view of the above findings, we hypothesized that induced expression of nestin in Müller glial cells or astrocytes may represent a reactive change of these cells to injury. In this connection, we sought to ascertain whether nestin expression would be induced in similar cells after hypoxia and, if so, whether the reactive change is reversible with hyperoxia treatment.

This study reports the expression of nestin, glutamine synthetase (GS), a marker for Müller glial cells, and GFAP, a marker for astrocytes, in Müller glial cells and astrocytes after hypoxia; furthermore, the induced expression of nestin was examined after post-hypoxia treatment. We demonstrate that although both Müller glial cells and astrocytes belong to macroglia in the retina, they exhibit differential response to the hypoxic insult, as judged by their response to the secondary hypoxia.

Materials and methods

Hypoxia and hyperoxia treatments

A total of 15 adult male Wistar rats (weighing 180–240 g; aged 8 weeks) were used in this study. Food and water were provided ad libitum, and the animals were maintained on a 12-h light–dark cycle. Five rats were used for normal study. The others (n = 10) were exposed to hypoxia by placing them in a hypoxia chamber (model 16M; Environmental Tectonics Corporation International, Southampton, PA, USA) at 9% O2 for 2 h. Of these, five rats were killed at 24 h following exposure, and the remaining five rats were immediately exposed to hyperoxia at 80% O2 for 2 h and then killed 24 h later (Kaur et al. 2005). In the handling and care of all animals, the International Guiding Principles for animal research, as stipulated by the Council for International Organizations of Medical Science (CIOMS) (1985) and as adopted by the Laboratory Animals 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

Following deep anaesthesia, the rats were perfused first with Ringer's solution followed by 4% paraformaldehyde in 0.1 m phosphate buffer (PB; pH 7.4). After perfusion, one eyeball of the respective rats was removed and postfixed in the same fixative for 2∼4 h before being 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 μm thickness and mounted on chrome-alum-gelatin-coated slides. The other eyeball was excised, and the entire retina was dissected out freely and postfixed in the same fixative for 1 h. The free retina was then processed for immunofluorescence analyses and flat-mounted.

Immunofluorescence

Frozen sections of the eye as well as free retinae were washed for 20 min in 0.01 m phosphate-buffered saline containing 0.1% Triton X-100 (PBS-T; pH 7.4) and blocked in 3% goat serum in PBS-T for 1 h. Double immunofluorescence labelling was carried out between nestin (1:500; Chemicon, CA, USA) and GS (1:1600; Chemicon), between nestin (1:500; Chemicon) and GFAP (1:1000; Dako corporation, CA, USA) and between GS (1:1600; Chemicon) and GFAP (1: 1000; Chemicon) antisera. The tissues were incubated in a medium containing a mixture of a rabbit polyclonal antiserum directed against GS or GFAP and a monoclonal mouse antiserum against nestin or GFAP in PBS-T overnight at room temperature. GS or GFAP (rabbit anti-rat) was visualized using fluoresceinisothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:200; Sigma, CA, USA). For nestin or GFAP (mouse anti-rat), cy3-conjugated goat anti-mouse IgG (1:200; Sigma) was used. After two rinses in the free retinae were mounted on chrome-alum-gelatin-coated slides and then coverslipped. Sections were examined in an Olympus confocal laser scanning microscope FV 1000.

Results

Nestin and GFAP expression in the retina subjected to hypoxia

In normal rats, GS immunoreactivity was localized mainly within the Müller glial cell soma and processes (arrows in Figure 1a). Some astrocytes in the ganglion cell layer (GCL) also stained positively for GS (Figure 1a). Nestin immunoreactivity was hardly detected and its labelling appeared to be associated mainly with some blood vessels (Figure 1b). GFAP antiserum specifically labelled astrocytes was confined exclusively to the GCL (Figure 1c). Nestin immunolabelling of some blood vessels was confirmed in the whole-mount free retina (arrows in Figure 1e,f). In the same preparation, double-labelling study revealed that astrocytes were devoid of nestin labelling in normal rats (Figure 1d–f). In rats subjected to hypoxia, it is clear that nestin was induced in astrocytes (arrows in Figure 2a–c) as well as the end-feet of Müller glial cells (arrowheads in Figure 2b,c) in whole-mount preparation. GFAP expression was absent in the end-feet of Müller glial cells (Figure 2c). In a sagittal section preparation, intense nestin expression was observed in the GCL in Müller glial cell processes colabelled with GS (Figure 2d–f). Many nestin-immunoreactive cells and tortuous processes appeared to span through all retinal layers with some processes extended as far as to the photoreceptor layer (arrows in Figure 2d–f). The induced nestin immunoreactivity was most pronounced in the GCL, where the retinal ganglion cell somata were enmeshed in a network formed by the astrocytic processes and end-feet of Müller glial cells. Nestin colabelled with GS indicated that the nestin-positive cells were Müller glial cells (arrows in Figure 2d–f). GFAP immunoexpression was not detected in Müller glial cells, and it remained relatively unchanged in the GCL compared to that in normal retina (Figure 2g–i).

Figure 1.

Figure 1

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The expression of nestin, GFAP and GS in normal rat retina. In normal rat retinae, GS immunoreactivity is localized mainly within the Müller glial cell soma and processes (arrows in a); some astrocytes in the GCL are also positively stained for GS (a). Nestin-positive cells are hardly detected except for some that are associated with the blood vessels (b). Anti-GFAP serum labels specifically astrocytes in the GCL (c). In the whole-mount preparation, GFAP immunoreactivity is localized mainly in astrocytes with radiating processes. The cells are evenly distributed in the GCL as viewed from the surface (d). The end-feet of Müller glial cells are devoid of GFAP immunoreactivity as shown by the clear background (d). Nestin immunostaining is absent in astrocytes and Müller glial cells (e,f), but some blood vessels are delineated by positive staining (arrows in e,f). Scale bar = 50 μm in f (applies to a–f). GCL, ganglion cell layer; GFAP, glial fibrilary acidic protein; GS, glutamine synthetase; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer.

Figure 2.

Figure 2

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The expression of nestin, GFAP and GS in rat retina subjected to hypoxia. Rats exposed to hypoxia for 2 h and killed at 24 h. In whole-mount preparation, it is clear that nestin is induced in astrocytes (arrows in a,b,c) as well as the end-feet of Müller glial cells (arrowheads in b,c). GFAP expression is absent in the end-feet of Müller glial cells (c). In a sagittal section preparation, many nestin-immunoreactive cells and processes appear to span through all retinal layers, including the ONL; some processes extended as far as to the photoreceptor layer (arrows in d,e,f). The increase in nestin immunoreactivity is most pronounced in the GCL, where astrocyte processes and end-feet of the Müller glial cells were intertwined. Nestin is colabelled with GS confirming that nestin-positive cells are Müller glial cells (arrows in d–f). There is no detectable GFAP expression in Müller glial cells, and it remains relatively stable in the GCL compared to that in normal retina (g–i). Scale bar = 50 μm in i (applies to a–i). GCL, ganglion cell layer; GFAP, glial fibrilary acidic protein; GS, glutamine synthetase; ONL, outer nuclear layer.

Nestin and GFAP expression in the retina subjected to hypoxia followed by hyperoxia treatment

In the retina of rats subjected to hypoxia for 2 h followed by hyperoxia for the same duration and killed at 24 h, nestin expression in the GCL was markedly attenuated. Nestin immunostaining was localized mainly in the GCL in a few fine processes of Müller glial cells (Figure 3a,b,c). In whole-mount preparation of the retina, nestin expression induced by hypoxia was retained in astrocytes (arrows in Figure 3d–f) but had diminished in the end-feet of Müller glial cells (Figure 3e,f).

Figure 3.

Figure 3

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The expression of nestin, GFAP and GS in rat retina subjected to hypoxia and followed by hyperoxia treating. Retinae of rats subjected to hypoxia for 2 h followed by immediate hyperoxia treatment for 2 h and killed at 24 h. Nestin expression in the GCL is markedly decreased. Nestin immunostaining is localized mainly in GCL and only in a few processes of Müller glial cells (a–c)). In whole-mount retinae, induced nestin expression is localized in astrocytes (arrows in e,f) but not in the end-feet of Müller glial cells (e,f). Scale bar = 50 μm in f (applies to a–f). GCL, ganglion cell layer; GFAP, glial fibrilary acidic protein; GS, glutamine synthetase.

Discussion

The retina, an extension of the CNS into the eye, does not enjoy the same degree of mechanical protection as the brain or spinal cord. It contains two major types of macroglial cells, namely, astrocytes, which are confined to the GCL, and Müller glial cells, whose processes span the entire thickness of the retina. The unique topographical distribution and configuration of Müller glial cells and astrocytes suggest that both glial types provide specific structural support for the retina (Bignami & Dahl 1979; Ekstrom et al. 1988). The retinal astrocytes reside in the domain of GCL and appear to associate more closely with the vasculature than with the ganglion cells and axons (Scherer & Schnitzer 1991; Huxlin et al. 1995). It is noteworthy that the glial sheaths encircling the somata of ganglion cells are formed predominantly by Muller glial cells (Hollander et al. 1991), which are known to express variable amounts of intermediate filaments (Lendahl et al. 1990; Huxlin et al. 1995).

It has been reported that astrocytes may express three types of intermediate filament proteins: GFAP, vimentin and nestin (Eliasson et al. 1999). Nestin and vimentin are the main intermediate filament proteins in immature astroglial cells, whereas maturing and adult astrocytes contain vimentin and GFAP (Eliasson et al. 1999). More recently, nestin has been demonstrated in adult reactive astrocytes following kainic acid lesions in the hippocampus and in direct spinal cord trauma without apparent reversion to the immature state after extended post-operative periods (Frisen et al. 1995; Clarke et al. 1999). Under normal circumstances, mature Müller glial cells do not express GFAP and nestin; however, in pathological conditions such as diabetic retinopathy(Zeng et al. 2000), glaucoma (Wang et al. 2000), ischaemia (Zahir et al. 2005), light damage (Heins & Aebi 1996) and retinal detachment (Grosche et al. 1995) GFAP can be induced in Müller glial cells. Recent studies have shown that nestin or transitin, a nestin-related intermediate filament, can be induced in retinal Müller glial cells following a toxic injury (Ooto et al. 2004; Fischer & Oma 2005) or laser injury (Kohno et al. 2006) in the adult retina. We reported recently that nestin and GFAP could be induced in Müller glial cells after optic transection and experimental glaucoma (Xue et al. 2006)) and proposed that nestin as well as GFAP is a sensitive marker for retinal injury. This notion is supported by the present observation of enhanced nestin expression in many, but not all, reactive astrocytes is seen following hypoxia treatment. A novel finding was the induced nestin expression in the Müller glial cells. On the other hand, unlike in the astrocytes, GFAP expression was not induced in Müller glial cells.

The induced expression of nestin in both reactive astrocytes and Müller glial cells suggests a structural plasticity of the cytoskeletal framework of these cells. Such a plastic change may be related to important environmental cues that may activate specific genes involved in metabolism and growth factor production to enhance functional recovery following hypoxia.

Another major finding in this study was the differential expression of nestin in astrocytes and Müller glial cells after hypoxia followed by hyperoxia treatment. In this group of rats, nestin immunostaining in Müller glial cells was downregulated to almost normal levels after hyperoxia. Remarkably, nestin immunofluorescence remained to be colabelled with GFAP in astrocytes; hence, its induced expression is sustained. A possible explanation for this may be that hypoxic insult has caused damage not only to retinal vasculature but also to ganglion cells resulting in induced nestin expression in both astrocytes and Müller glial cells. Whereas injury to the ganglion cells can be reversed by immediate hyperoxia treatment, present results suggest that this may not follow in the retinal vasculature. In view of the close spatial relationship of Müller glial cells specifically with the ganglion cells, while astrocytes with the retinal vasculature, it is therefore not unreasonable to suggest that this may have associated for the differential nestin expression with hyperoxia treatment.

Nestin is a plastic cytoskeleton protein whose expression may vary with the functional states of the cells. The above speculation takes into consideration the possibility that hyperoxia could have afflicted more severe damage to the retinal vasculature so that nestin expression in astrocytes that are closely associated with them both structurally and functionally remained elevated despite hyperoxia treatment. Clinically, hyperoxia has been used to treat hypoxic retinal degeneration. It is well documented that both hypoxia and hyperoxia can cause tissue stress and damage (Yu & Cringle 2002). The results obtained from the present animal model of retina hypoxia/hyperoxia may provide relevant information for improving therapeutic optimum oxygen intervention strategies for retinal degeneration.

Acknowledgments

The authors thank Professor Ling Eng-Ang (Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore) for his helpful suggestions. This work was funded from National Science Foundation of Yun Nan province (No.2009C135).

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