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Signs of Müller cell gliotic response found in the retina of newts exposed to real and simulated microgravity
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Advances in Space Research 49 (2012) 1465–1471 www.elsevier.com/locate/asr
Signs of Mu¨ller cell gliotic response found in the retina of newts exposed to real and simulated microgravity E.N. Grigoryan a,⇑, H.J. Anton b, V.A. Poplinskaya a, K.S. Aleinikova a, E.I. Domaratskaya a, Y.P. Novikova a, E. Almeida c a
Kol’tsov Institute of Developmental Biology, RAS, Vavilov str. 26, 119991 Moscow, Russia b Institute of Zoology, Cologne University, Weyertal 119, D 50923 Ko¨ln, Germany c NASA Ames Research Center, Moffett Field, CA, USA
Received 25 August 2010; received in revised form 7 February 2012; accepted 22 February 2012 Available online 5 March 2012
Abstract The effects of real and simulated microgravity on the eye tissue regeneration of newts were investigated. For the first time changes in Mu¨ller glial cells in the retina of eyes regenerating after retinal detachment were detected in newts exposed to clinorotation. The cells divided, were hypertrophied, and their processes were thickened. Such changes suggested reactive gliosis and were more significant in animals exposed to rotation when compared with desk-top controls. Later experiments onboard the Russian biosatellite Bion-11 showed similar changes in the retinas that were regenerating in a two-week spaceflight. In the Bion-11 animals, GFAP, the major structural protein of retinal macroglial cells, was found to be upregulated. In a more recent experiment onboard Foton-M3 (2007), GFAP expression in retinas of space-flown, ground control (kept at 1 g), and basal control (sacrificed on launch day) newts was quantified, using microscopy, immunohistochemistry, and digital image analysis. A low level of immunoreactivity was observed in basal controls. In contrast, retinas of space-flown animals showed greater GFAP immunoreactivity associated with both an increased cell number and a higher thickness of intermediate filaments. This, in turn, was accompanied by up-regulation of stress protein (HSP90) and growth factor (FGF2) expressions. It can be postulated that such a response of Mu¨ller cells was to mitigate the retinal stress in newts exposed to microgravity. Taken together, the data suggest that the retinal population of macroglial cells could be sensitive to gravity changes and that in space it can react by enhancing its neuroprotective function. Ó 2012 COSPAR. Published by Elsevier Ltd. All rights reserved. Keywords: Newt; Retina; Gliosis; Mu¨ller cells; Spaceflight; Simulated microgravity
1. Introduction Understanding the pattern of regeneration processes in animals and humans exposed to the space environment is of both theoretical and practical importance: study of animal tissue restoration can help identify the phenomena that can be extrapolated to humans. Our newt Pl. waltl (Urodela) experiments demonstrated that exposure to the space environment stimulated cell pro⇑ Corresponding author. Tel.: +7 495 6085679; fax: +7 499 1358012.
E-mail addresses: [email protected] (E.N. Grigoryan), [email protected] (H.J. Anton), [email protected] (E. Almeida).
liferation as well as lens, limb and tail regeneration (Mitashov et al., 1987, 1996; Grigoryan et al., 2002). Fast rotating clinostat studies showed that cellular processes involved in tissue regeneration (cell proliferation, migration, differentiation) developed at a higher rate and occurred in a more synchronized way in simulated microgravity when compared to 1 g controls (Anton et al., 1996). Results of real and simulation spaceflight experiments taken together with the data about newt habitats and behavior led us to believe that animal pre-adaptation to a lower gravity field (buyoncy) facilitated tissue regeneration in space. One of the processes involved in newt eye regeneration is a retinal glial cell (macroglial, Mu¨ller cell) response. We
0273-1177/$36.00 Ó 2012 COSPAR. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.asr.2012.02.025
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observed it in laboratory animals used in in vivo retinal detachment experiments (Novikova et al., 2008) as well as in isolated retinal cultures grown in vitro by 3D rotation (Grigoryan, 2007; Novikova et al., 2010). To-date, there are no publications covering the effects of microgravity or other spaceflight factors on the glial population in the animal and human retina. It is however recognized that macroglia plays a very important part in the retinal function (Newman and Reichenbach, 1996) and repair following its lesion (Dyer and Cepko, 2000; Lewis and Fisher, 2003). Population of Mu¨ller cells provide scaffolding across the neural retina and are in continuous contact with retinal neurons via neurotransmitters, [Ca2+] and ATP (Metea and Newman, 2006). It is also known that the state of the retinal macroglia is a good indicator of eye health, which changes in response to various stimulations. Its changes are involved in such eye diseases as retinal detachment (Erickson et al., 1987), proliferative retinopathy (Wickham and Charteris, 2009), and macular dystrophy (Wu et al., 2003). It is understood that Mu¨ller cells can be affected by elevated intraocular pressure (Lam et al., 2003; Woldemussie et al., 2004), mechanical tension (Lindqvist et al., 2010), ischemia (Kuhrt et al., 2008), or fluid-electrolyte metabolism changes (Qin et al., 2009). In response to retinal injury, Mu¨ller cells undergo changes some of which help to protect the retina from its further deterioration. These cells produce growth factors, antioxidants, and neurotransmitters (Garcı´a and Vecino, 2003). Moreover, in some adult vertebrates Mu¨ller cells can serve as sources of undifferentiated precursors (Jadhav et al., 2009). In our flight and simulation experiments we frequently observed microgravity-induced changes in Mu¨ller cells in the intact (non-operated) and regenerating retina. The present paper summarizes the data giving evidences that gravitational changes in space or on the ground may cause an increase in the macroglial population as well as an enhancement of its GFAP (glial fibrillar acidic protein) expression. In space-flown newts these shifts are correlated with HSP90 (generalized stress protein) and FGF2 (growth factor) up-regulation. 2. Materials and methods 2.1. Bion-11 experiment In this and all subsequent experiments we used mature Pl. waltl newts grown in the aquarium facility of the Institute of Developmental Biology, Russian Academy of Sciences (RAS). The animals were kept and operated in strict adherence to the RAS rules of animal care and use. According to the experiment protocol, the optic nerve and blood vessels of the flight, basal (sacrificed on launch day), aquarium, and synchronous control animals were cut bilaterally two or four weeks prior to launch (Mitashov, 1970). Each group included 6 animals. On launch
day eyes of 2 newts from the groups were fixed in Bouin’s solution and 4% PFA. The animals of space-flight groups operated 2 and 4 weeks before launch were housed in a Newt container and flown for 14 days onboard Bion-11. A day after launch a synchronous control experiment was started. The experimental protocol was described in great detail earlier (Grigoryan et al., 1999). Eyes of the flight, basal and synchronous control newts fixed in Bouin’s solution were exposed to morphological examinations, and those fixed in 4% PFA were frozen in liquid nitrogen, sectioned and stained immunohistochemically with antibodies against GFAP (Sigma) marking the retinal macroglia of newts and other animals. Visualization was performed using secondary FITC-labeled antibodies (IgG, Sigma). Immunospecific staining was analyzed in an Olympus H3 microscope, and images were recorded at identical magnifications and exposure times to allow correct comparison of the fluorescence intensity (GFAP expression). Reaction specificity was identified using the sections treated by secondary antibodies in the absence of the first ones. 2.2. Clinostat experiment simulating microgravity effect To induce reparative processes, the retina was detached from the pigment epithelium, according to the previously developed procedure (Grigorian et al., 1990). The procedure was performed on both eyes of 16 newts 9 days before clinostating. Half of the experimental animals were later rotated in a vertically-oriented clinostat at the Zoological Institute, Cologne University. The clinostating at 60 rpm continued for 7 days in a dark room at ambient temperature and 100% humidity. These environmental parameters were close to those in the space experiment. During clinostating the animals experienced 10 3 – 10 2 g, as measured according to Silver (1976). Immediately after clinorotation and 7 days later the eyes were fixed in Bouin’s solution and treated using routine histology techniques. Synchronous controls were kept on a blanket in an aquarium containing a small amount of water. All other environmental parameters were the same as in the clinostat experiment. Synchronous biosamples were fixed similarly to the clinostat specimens. Immediately after the experiment two animals from each group were injected with DNA precursor [3H]TdR (with specific activity of 4.6 Ci/mM, 5 mCi/g) to detect retinal cell proliferation. Histological samples, semi-thin sections and radioautographs were obtained using standard procedures. Retinal morphological parameters as well as the number and localization of proliferating cells were identified in eye cross-sections (5 lm). The number of accessory prolongations of Mu¨ller cells was measured in every other section of a complete set separately in the central and peripheral sites after fast red staining with the microscope diaphragm halfclosed using NS-9 and ZS-1 filters (Russian Standard 9411-60). The resultant data were statistically processed using Excel software provided that the number of
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examined eyes was at least 6 and that of sections no less than 100. 2.3. Foton-M3 experiment Ten days prior to launch, basal controls (7 newts) and flight newts (16 newts) underwent surgery. The corneas of both eyes were incised transversally and lenses were removed. Two days later synchronous controls (16 newts) underwent the same surgical procedures. The Foton-M3 flight continued for 12 days. On the launch day, basal controls were treated to study eye regeneration immediately prior to flight. Regenerating eyes of the newts from flight, basal and synchronous groups were fixed for further morphological, immunochemical and molecular examinations. Eyes fixed in Bouin’s solution were treated according to routine histological procedures, and serial sections (6 lm) stained with hematoxylin-eosin were prepared. Eyes fixed in 4% PFA were washed in sucrose on phosphate buffer and frozen in a medium (Jung, Leica Instruments GmbH) in liquid nitrogen. Then frozen sections (10 lm) were prepared and stained by standard immunochemical procedures. To do that, commercial monoclonal antibodies against GFAP (1:100), heat shock protein 90 (1:400), as well as rabbit antibodies anti-human fibroblast growth factor basic (FGFb) (all Sigma) at 1:100 were used. Preparations stained with the secondary antibody (FITCconjugated, anti-mouse, 1:50) were analyzed in an Olympus H3 microscope and images were recorded using a Lite software packet. Using software Adobe Photoshop CS3 Extended, fluorescence staining intensity in the inner retina was measured in all groups of animals. This was done with respect to the parameter: mean of stained area (in pixels) as a percent of randomly selected fields (in pixels, 20 for each group) in the images of central sites of the retinas of two eyes from the animals of four groups (intact newts, basal control, synchronous control, and “flown” animals. 3. Results 3.1. Bion-11 experiment This experiment was performed on newts with the optic nerve cut two or 4 weeks prior to launch. During the orbital flight a new retina was formed through the stages we described earlier (Grigoryan et al., 1999). When regenerated, all retinal layers and cell types, including glial cells, developed differentiation. Using immunocytochemical methods with specific anti-GFAP antibodies, we detected GFAP+ cells at the onset of differentiation, the number of which increased as the regeneration progressed. The first GFAP+ cells were observed at stages IV–V (i.e., 4 weeks after surgery), and the cell population at stage VI (6 weeks after surgery) of the retinal regeneration. The earliest cells showing specific immune response were blast cells of the retina anlage as well as cells in the vicinity of the eye growth zone (ora serrata). This indicated that GFAP
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expression was widely spread occurring in the cells beyond the location of prospective Mu¨ller cells. In order to describe differentiating Mu¨ller cells (stage VI) in the space-flown and synchronous newts, we measured fluorescence intensity and counted the GFAP+ cells. It turned out that both the fluorescence intensity and the number of GFAP+ cells were greater in the eye regenerates of the flight newts. GFAP expression in the flight animals was by 90% and in the synchronous controls by 60% higher than the baseline. The number of GFAP+ cells in the retinal regenerates of the flight newts was by 40% higher than in those of the controls. This suggested that the space environment caused changes in the glial population of the retina regenerating on orbit. These changes as well as those described below included both an increase in the number of Mu¨ller cells and an enhancement of protein expression of their intermediate filaments – GFAP. 3.2. Clinostat experiment simulating microgravity effect Morphological examination of the eyes immediately after retinal detachment showed that the retina was separated from the pigment epithelium though the number of dead cells was low (Fig. 1A). When clinostating was stopped (16 days after surgery), the detached retina contained a minor pool of [3H]-TdR+ cells, most of which were located in the inner nuclear layer (INL). This observation held true for synchronous controls. The number of proliferating cells in the central area was greater than in the periphery. Examination of semi-thin (1 lm) sections showed that the cells containing [3H]-TdR nuclei belonged to the Mu¨ller cell population (Fig. 1B). Single [3H]-TdR nuclei were also seen in the outer nuclear layer (ONL). We found that the absolute number of proliferating Mu¨ller cells in the detached retina of the rotated newts exceeded that in the controls, the difference being most significant in the INL of the central part of the eye cup (Fig. 2A). Examination of the number of accessory prolongations of Mu¨ller cells in the inner plexiform layer of different retinal areas indicated a significant increase of the macroglial population immediately as well as 7 days after clinorotation (Fig. 2B). The more noticeable increase in the number of accessory prolongations of Mu¨ller cells in the retina of the newts from both groups was detected in its center, as was the case with an increase in the count of DNA-synthesizing cells. In summary, gravitational changes enhanced the proliferation of Mu¨ller cells caused by the retinal detachment, which resulted in an increased number of their accessory prolongations when compared to the controls. In other words, the gliotic response in the detached retina was essentially reinforced by clinorotation. 3.3. Foton-M3 experiment In this experiment we used Wolfian regeneration model and investigated lens repair (see Grigoryan et al., 2008) as well as GFAP expression in the Mu¨ller cell population and
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Fig. 1. Retinal detachment in the experiments used physiological weightlessness simulated by clinorotation. (A) - view of the newt’s eye after retinal detachment. Bar: 500 lm; (B) –Semithin section (1 l) of individual Mu¨ller glial cell with [3H]-TdR labeled nuclei (arrow).
Fig. 2. Increase of cell population of Mu¨ller glia in the retina of clinorotated animals as compared with controls at the 16th day after retinal detachment and 7th day of rotation. (A) - Absolute number of [3H]-TdR labeled cells in the central part of the retina in rotated (dark column) and control (light column) animals. ONL - retinal outer nuclear layer, INL – inner nuclear layer, GCL - ganglion cell layer. (B) - Relative number (per one section, M ± m) of Mu¨ller glial cell processes in different zones of the retina in rotated (dark column) and control (light column) animals. Dors – dorsal, Centr – central, Ventr – ventral retinal regions. An increase of a number of [3H]-TdR labeled cells in INL (area of Mu¨ller cell bodies’ location) correlates with the increase of a number of Mu¨ller glial cell processes at the peripheral and central retina.
FGF2 and HSP90 expression in the eyes of space-flown and synchronous control newts. Using monoclonal antibodies, FGF2 expression playing the key role in the eye development and regeneration was examined by immunohistochemical methods. Sections from all animal groups (basal control, flight, synchronous control) were treated simultaneously following the same protocol, and images were obtained after the same exposure time. In basal controls that were at stage II of lens regeneration, immunospecific fluorescence was detected in the retinal interior and in the growth zone. In synchronous controls, that were at stage VII, and in the flight animals, that were at stage VIII, growth zone cells were also stained, some cells showing more intense staining at their periphery. Both flight and synchronous animals showed fluorescence in the iris vascular membrane, cornea epithelium and eye growth zone. However, the intensity of FGF2 expression in the flight animals was higher than in the controls (Fig. 3A and B). Using immunochemistry methods, we also investigated the expression of HSP90 heat shock protein as another potential regulator of eye regeneration in space. We found for the first time that antibodies against the protein were most actively bound to retinal Mu¨ller cell. In basal controls, the reaction, although weak, was sufficient to identify immunofluorescence localization. We detected it in the perikaria and accessory prolongations of glial cells, as well
as in the photoreceptor layer and in the retina, where it was less visible. On the whole, the staining intensity (or fluorescence level) was greater in the sections of regenerating eyes of the flight newts compared to that in basal and synchronous controls. The fluorescence level was the highest at the site of expanding end-feet of accessory prolongations of glial cell (Fig. 4A and B). Moreover, Mu¨ller cell processes in the outer retina of the flight animals also showed a distinct immune reaction. The staining intensity of glial cells of the regenerating eyes of synchronous controls was significantly lower, reaching the level between that in basal controls and in flight animals. In Foton-M3 experiment, GFAP expression in retinas of space-flown, ground control (kept at 1 g), and basal control newts was quantified using microscopy, immunohistochemistry, and digital image analysis. It was found that Mu¨ller cell processes of nonoperated animals displayed relatively low GFAP immunolabeling. A low level of immunoreactivity was also observed in basal controls. In contrast, retinas of spaceflown animals showed greater GFAP immunoreactivity associated with both an increased cell number and a higher density of intermediate filaments. Using custom designed software, we measured means of the stained area (in pixels) as a percent of the entire field (in pixels). Random fields of the same size were marked in the images of central sites of eye retinas isolated from the newts of all three groups and
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Fig. 3. Expression of fibroblast growth factor (FGFb) in the eye vascular network (arrows) of “flown” (A) and synchron (B) newts studied soon after landing of Foton M-3. Bar: 20 lm. In the presented case immuno-fluorescence intensity evaluated by means of estimation of equally stained areas (tolerance = 40) was: A – 3167 and B – 5321 pixels.
Fig. 4. Expression of heat shock protein 90 (HSP 90) in the retinal Mu¨ller cell endfeet (arrows) of retinal Mu¨ller cells in flight (A) and synchron (B) animals. GL – ganglion cell layer, IPL – inner plexiform layer, INL – inner nuclear layer of the retina. Bar: 20 lm.
group of intact animals. As can be seen in Fig. 5, the parameter was greater in the flight animals when compared to the controls. 4. Discussion The results of newt experiments onboard Bion-11 and Foton-M3 as well as in clinorotation demonstrated that the gliotic response of the retinal macroglial population was stimulated by an altered gravitational field. During clinorotation the proliferation of Mu¨ller cells and the number of their accessory prolongations in the detached retina increased when compared to the controls. This phenomenon manifested a response of the macroglial population to the shift in retinal physiology initially caused by the ret-
Fig. 5. Computer data of relative intensity of GFAP expression in the square unit in the central area of the retina of intact newts (black), basal control (white), synchronous control (light grey) and flight (dark grey column) animals.
ina detachment and subsequently enhanced by clinorotation. With respect to the parameters we measured, the most significant differences were detected in the central retina. It is important to note here that this is the area where serious eye disorders, e.g., macular degeneration, develop (Nowak, 2006). The Bion-11 experiment, in which the retina was regenerating after the optic nerve was cut, revealed changes in the glial population, viz., GFAP upregulation assumed to be an indicator of retinal stress. For instance, in response to the retinal detachment or bright light damage the concentrations of GFAP, Mu¨ller cells and their prolongations increase. This type of reaction is observed in response to different retinal injuries and in various animal classes. Study of GFAP expression in the retina of newts, an animal model used in space-flown regeneration experiments, may provide insight into the role the Mu¨ller glia plays in an altered gravitational environment. Examination of the Mu¨ller cell population in the FotonM3 experiment gave support to the observation concerning glial response enhancement in the flight newts compared to the controls. Similarly to what was found in previous flights, GFAP expression was up-regulated when measured by immunospecific fluorescence intensity. However, in the Foton-M3 newts GFAP up-regulation developed together with FGF2 and HSP90 up-regulation. It was previously demonstrated that FGF2 was involved in the induction and regulation of cell proliferation as well as in the expres-
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sion of proteins of intermediate filaments of Mu¨ller cells (Lewis et al., 1992). Moreover, it was suggested that the phenotype changes of Mu¨ller cells could be induced by exogenous growth factors in the absence of retinal damage (Fisher et al., 2004). From our perspective, it is important to consider potential interactions of FGF2 and HSPs as well as HSPs and GFAP. The HSP90 presence in the regenerating eye retina and particularly in Mu¨ller cells suggests that this protein can be engaged in the Mu¨ller cell gliotic response. HSP expression in the same cell population in response to a stress was demonstrated earlier both in vivo (Gohdo et al., 2001) and in vitro (Hauck et al., 2003). We believe that an enhancement of the Mu¨ller cell gliotic response we detected in three different experiments using different methods of retinal injury was not induced by gravity changes per se but was mediated by changes in the whole organism. There are observations indicating intraocular pressure (IOP) changes in humans exposed to simulated microgravity (Kuzmin, 1984), real space missions (Schwartz et al., 1993) as well as to rapidly changing g levels (Kroll et al., 1987). However, rats subjected to laser-induced IOP elevation showed loss of some ganglion cells which was followed by a gradual and sustained increase in GFAP immunoreactivity (Lam et al., 2003). Another cause of up-regulated gliotic response to the microgravity environment may be ion balance changes in the eye vitreous body. As known, variations in the internal environment (including ion composition) of the retinal radial glial cells can modify their function and the spectrum of molecules they synthesize (Steinbach and Schlosshauer, 2000). On the whole, the phenomenon is aimed at retina protection. In conclusion, adequate understanding of the signaling mechanisms implicated in gliotic alterations of Mu¨ller cells in the space environment is essential for preventing excessive development and destructive effects of the glia in the retina. References Anton, H.J., Grigoryan, E.N., Mitashov, V.I. Influence of longitudinal whole animal clinorotation on lens, tail, and limb regeneration in Urodeles. Adv. Space Res. 17, 55–65, 1996. Dyer, M.A., Cepko, C.L. Control of Mu¨ller glial cell proliferation and activation following injury. Nat. Neurosci. 3, 873–880, 2000. Erickson, P.A., Fisher, S.K., Gue´rin, C.J., Anderson, D.H., Kaska, D.D. Glial fibrillary acidic protein increases in Mu¨ller cells after retinal detachment. Exp. Eye Res. 44, 37–48, 1987. Fisher, A.J., Omar, G., Eubanks, J., McGuire, Ch.R., Dierks, B.D., Reh, T.A. Different aspects of gliosis in retinal Mu¨ller glia can be induced by CNTF, insulin, and FGF2 in the absence of damage. Mol. Vis. 10, 973–986, 2004. Garcı´a, M., Vecino, E. Role of Mu¨ller glia in neuroprotection and regeneration in the retina. Histol. Histopathol. 18, 1205–1218, 2003. Gohdo, T., Ueda, H., Ohno, S., Iijima, H., Tsukahara, S. Heat shock protein 70 expression increased in rabbit Mu¨ller cells in the ischemiareperfusion model. Ophthalm. Res. 33, 298–302, 2001. Grigoryan, E.N. Alternative intrinsic cell sources for neural retina regeneration in adult urodelean amphibians, in: Chiba, Ch. (Ed.), Strategies for Retinal Tissue Repair and Regeneration in Vertebrates:
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Advances in Space Research 49 (2012) 1465–1471 www.elsevier.com/locate/asr
Signs of Mu¨ller cell gliotic response found in the retina of newts exposed to real and simulated microgravity E.N. Grigoryan a,⇑, H.J. Anton b, V.A. Poplinskaya a, K.S. Aleinikova a, E.I. Domaratskaya a, Y.P. Novikova a, E. Almeida c a
Kol’tsov Institute of Developmental Biology, RAS, Vavilov str. 26, 119991 Moscow, Russia b Institute of Zoology, Cologne University, Weyertal 119, D 50923 Ko¨ln, Germany c NASA Ames Research Center, Moffett Field, CA, USA
Received 25 August 2010; received in revised form 7 February 2012; accepted 22 February 2012 Available online 5 March 2012
Abstract The effects of real and simulated microgravity on the eye tissue regeneration of newts were investigated. For the first time changes in Mu¨ller glial cells in the retina of eyes regenerating after retinal detachment were detected in newts exposed to clinorotation. The cells divided, were hypertrophied, and their processes were thickened. Such changes suggested reactive gliosis and were more significant in animals exposed to rotation when compared with desk-top controls. Later experiments onboard the Russian biosatellite Bion-11 showed similar changes in the retinas that were regenerating in a two-week spaceflight. In the Bion-11 animals, GFAP, the major structural protein of retinal macroglial cells, was found to be upregulated. In a more recent experiment onboard Foton-M3 (2007), GFAP expression in retinas of space-flown, ground control (kept at 1 g), and basal control (sacrificed on launch day) newts was quantified, using microscopy, immunohistochemistry, and digital image analysis. A low level of immunoreactivity was observed in basal controls. In contrast, retinas of space-flown animals showed greater GFAP immunoreactivity associated with both an increased cell number and a higher thickness of intermediate filaments. This, in turn, was accompanied by up-regulation of stress protein (HSP90) and growth factor (FGF2) expressions. It can be postulated that such a response of Mu¨ller cells was to mitigate the retinal stress in newts exposed to microgravity. Taken together, the data suggest that the retinal population of macroglial cells could be sensitive to gravity changes and that in space it can react by enhancing its neuroprotective function. Ó 2012 COSPAR. Published by Elsevier Ltd. All rights reserved. Keywords: Newt; Retina; Gliosis; Mu¨ller cells; Spaceflight; Simulated microgravity
1. Introduction Understanding the pattern of regeneration processes in animals and humans exposed to the space environment is of both theoretical and practical importance: study of animal tissue restoration can help identify the phenomena that can be extrapolated to humans. Our newt Pl. waltl (Urodela) experiments demonstrated that exposure to the space environment stimulated cell pro⇑ Corresponding author. Tel.: +7 495 6085679; fax: +7 499 1358012.
E-mail addresses: [email protected] (E.N. Grigoryan), [email protected] (H.J. Anton), [email protected] (E. Almeida).
liferation as well as lens, limb and tail regeneration (Mitashov et al., 1987, 1996; Grigoryan et al., 2002). Fast rotating clinostat studies showed that cellular processes involved in tissue regeneration (cell proliferation, migration, differentiation) developed at a higher rate and occurred in a more synchronized way in simulated microgravity when compared to 1 g controls (Anton et al., 1996). Results of real and simulation spaceflight experiments taken together with the data about newt habitats and behavior led us to believe that animal pre-adaptation to a lower gravity field (buyoncy) facilitated tissue regeneration in space. One of the processes involved in newt eye regeneration is a retinal glial cell (macroglial, Mu¨ller cell) response. We
0273-1177/$36.00 Ó 2012 COSPAR. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.asr.2012.02.025
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observed it in laboratory animals used in in vivo retinal detachment experiments (Novikova et al., 2008) as well as in isolated retinal cultures grown in vitro by 3D rotation (Grigoryan, 2007; Novikova et al., 2010). To-date, there are no publications covering the effects of microgravity or other spaceflight factors on the glial population in the animal and human retina. It is however recognized that macroglia plays a very important part in the retinal function (Newman and Reichenbach, 1996) and repair following its lesion (Dyer and Cepko, 2000; Lewis and Fisher, 2003). Population of Mu¨ller cells provide scaffolding across the neural retina and are in continuous contact with retinal neurons via neurotransmitters, [Ca2+] and ATP (Metea and Newman, 2006). It is also known that the state of the retinal macroglia is a good indicator of eye health, which changes in response to various stimulations. Its changes are involved in such eye diseases as retinal detachment (Erickson et al., 1987), proliferative retinopathy (Wickham and Charteris, 2009), and macular dystrophy (Wu et al., 2003). It is understood that Mu¨ller cells can be affected by elevated intraocular pressure (Lam et al., 2003; Woldemussie et al., 2004), mechanical tension (Lindqvist et al., 2010), ischemia (Kuhrt et al., 2008), or fluid-electrolyte metabolism changes (Qin et al., 2009). In response to retinal injury, Mu¨ller cells undergo changes some of which help to protect the retina from its further deterioration. These cells produce growth factors, antioxidants, and neurotransmitters (Garcı´a and Vecino, 2003). Moreover, in some adult vertebrates Mu¨ller cells can serve as sources of undifferentiated precursors (Jadhav et al., 2009). In our flight and simulation experiments we frequently observed microgravity-induced changes in Mu¨ller cells in the intact (non-operated) and regenerating retina. The present paper summarizes the data giving evidences that gravitational changes in space or on the ground may cause an increase in the macroglial population as well as an enhancement of its GFAP (glial fibrillar acidic protein) expression. In space-flown newts these shifts are correlated with HSP90 (generalized stress protein) and FGF2 (growth factor) up-regulation. 2. Materials and methods 2.1. Bion-11 experiment In this and all subsequent experiments we used mature Pl. waltl newts grown in the aquarium facility of the Institute of Developmental Biology, Russian Academy of Sciences (RAS). The animals were kept and operated in strict adherence to the RAS rules of animal care and use. According to the experiment protocol, the optic nerve and blood vessels of the flight, basal (sacrificed on launch day), aquarium, and synchronous control animals were cut bilaterally two or four weeks prior to launch (Mitashov, 1970). Each group included 6 animals. On launch
day eyes of 2 newts from the groups were fixed in Bouin’s solution and 4% PFA. The animals of space-flight groups operated 2 and 4 weeks before launch were housed in a Newt container and flown for 14 days onboard Bion-11. A day after launch a synchronous control experiment was started. The experimental protocol was described in great detail earlier (Grigoryan et al., 1999). Eyes of the flight, basal and synchronous control newts fixed in Bouin’s solution were exposed to morphological examinations, and those fixed in 4% PFA were frozen in liquid nitrogen, sectioned and stained immunohistochemically with antibodies against GFAP (Sigma) marking the retinal macroglia of newts and other animals. Visualization was performed using secondary FITC-labeled antibodies (IgG, Sigma). Immunospecific staining was analyzed in an Olympus H3 microscope, and images were recorded at identical magnifications and exposure times to allow correct comparison of the fluorescence intensity (GFAP expression). Reaction specificity was identified using the sections treated by secondary antibodies in the absence of the first ones. 2.2. Clinostat experiment simulating microgravity effect To induce reparative processes, the retina was detached from the pigment epithelium, according to the previously developed procedure (Grigorian et al., 1990). The procedure was performed on both eyes of 16 newts 9 days before clinostating. Half of the experimental animals were later rotated in a vertically-oriented clinostat at the Zoological Institute, Cologne University. The clinostating at 60 rpm continued for 7 days in a dark room at ambient temperature and 100% humidity. These environmental parameters were close to those in the space experiment. During clinostating the animals experienced 10 3 – 10 2 g, as measured according to Silver (1976). Immediately after clinorotation and 7 days later the eyes were fixed in Bouin’s solution and treated using routine histology techniques. Synchronous controls were kept on a blanket in an aquarium containing a small amount of water. All other environmental parameters were the same as in the clinostat experiment. Synchronous biosamples were fixed similarly to the clinostat specimens. Immediately after the experiment two animals from each group were injected with DNA precursor [3H]TdR (with specific activity of 4.6 Ci/mM, 5 mCi/g) to detect retinal cell proliferation. Histological samples, semi-thin sections and radioautographs were obtained using standard procedures. Retinal morphological parameters as well as the number and localization of proliferating cells were identified in eye cross-sections (5 lm). The number of accessory prolongations of Mu¨ller cells was measured in every other section of a complete set separately in the central and peripheral sites after fast red staining with the microscope diaphragm halfclosed using NS-9 and ZS-1 filters (Russian Standard 9411-60). The resultant data were statistically processed using Excel software provided that the number of
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examined eyes was at least 6 and that of sections no less than 100. 2.3. Foton-M3 experiment Ten days prior to launch, basal controls (7 newts) and flight newts (16 newts) underwent surgery. The corneas of both eyes were incised transversally and lenses were removed. Two days later synchronous controls (16 newts) underwent the same surgical procedures. The Foton-M3 flight continued for 12 days. On the launch day, basal controls were treated to study eye regeneration immediately prior to flight. Regenerating eyes of the newts from flight, basal and synchronous groups were fixed for further morphological, immunochemical and molecular examinations. Eyes fixed in Bouin’s solution were treated according to routine histological procedures, and serial sections (6 lm) stained with hematoxylin-eosin were prepared. Eyes fixed in 4% PFA were washed in sucrose on phosphate buffer and frozen in a medium (Jung, Leica Instruments GmbH) in liquid nitrogen. Then frozen sections (10 lm) were prepared and stained by standard immunochemical procedures. To do that, commercial monoclonal antibodies against GFAP (1:100), heat shock protein 90 (1:400), as well as rabbit antibodies anti-human fibroblast growth factor basic (FGFb) (all Sigma) at 1:100 were used. Preparations stained with the secondary antibody (FITCconjugated, anti-mouse, 1:50) were analyzed in an Olympus H3 microscope and images were recorded using a Lite software packet. Using software Adobe Photoshop CS3 Extended, fluorescence staining intensity in the inner retina was measured in all groups of animals. This was done with respect to the parameter: mean of stained area (in pixels) as a percent of randomly selected fields (in pixels, 20 for each group) in the images of central sites of the retinas of two eyes from the animals of four groups (intact newts, basal control, synchronous control, and “flown” animals. 3. Results 3.1. Bion-11 experiment This experiment was performed on newts with the optic nerve cut two or 4 weeks prior to launch. During the orbital flight a new retina was formed through the stages we described earlier (Grigoryan et al., 1999). When regenerated, all retinal layers and cell types, including glial cells, developed differentiation. Using immunocytochemical methods with specific anti-GFAP antibodies, we detected GFAP+ cells at the onset of differentiation, the number of which increased as the regeneration progressed. The first GFAP+ cells were observed at stages IV–V (i.e., 4 weeks after surgery), and the cell population at stage VI (6 weeks after surgery) of the retinal regeneration. The earliest cells showing specific immune response were blast cells of the retina anlage as well as cells in the vicinity of the eye growth zone (ora serrata). This indicated that GFAP
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expression was widely spread occurring in the cells beyond the location of prospective Mu¨ller cells. In order to describe differentiating Mu¨ller cells (stage VI) in the space-flown and synchronous newts, we measured fluorescence intensity and counted the GFAP+ cells. It turned out that both the fluorescence intensity and the number of GFAP+ cells were greater in the eye regenerates of the flight newts. GFAP expression in the flight animals was by 90% and in the synchronous controls by 60% higher than the baseline. The number of GFAP+ cells in the retinal regenerates of the flight newts was by 40% higher than in those of the controls. This suggested that the space environment caused changes in the glial population of the retina regenerating on orbit. These changes as well as those described below included both an increase in the number of Mu¨ller cells and an enhancement of protein expression of their intermediate filaments – GFAP. 3.2. Clinostat experiment simulating microgravity effect Morphological examination of the eyes immediately after retinal detachment showed that the retina was separated from the pigment epithelium though the number of dead cells was low (Fig. 1A). When clinostating was stopped (16 days after surgery), the detached retina contained a minor pool of [3H]-TdR+ cells, most of which were located in the inner nuclear layer (INL). This observation held true for synchronous controls. The number of proliferating cells in the central area was greater than in the periphery. Examination of semi-thin (1 lm) sections showed that the cells containing [3H]-TdR nuclei belonged to the Mu¨ller cell population (Fig. 1B). Single [3H]-TdR nuclei were also seen in the outer nuclear layer (ONL). We found that the absolute number of proliferating Mu¨ller cells in the detached retina of the rotated newts exceeded that in the controls, the difference being most significant in the INL of the central part of the eye cup (Fig. 2A). Examination of the number of accessory prolongations of Mu¨ller cells in the inner plexiform layer of different retinal areas indicated a significant increase of the macroglial population immediately as well as 7 days after clinorotation (Fig. 2B). The more noticeable increase in the number of accessory prolongations of Mu¨ller cells in the retina of the newts from both groups was detected in its center, as was the case with an increase in the count of DNA-synthesizing cells. In summary, gravitational changes enhanced the proliferation of Mu¨ller cells caused by the retinal detachment, which resulted in an increased number of their accessory prolongations when compared to the controls. In other words, the gliotic response in the detached retina was essentially reinforced by clinorotation. 3.3. Foton-M3 experiment In this experiment we used Wolfian regeneration model and investigated lens repair (see Grigoryan et al., 2008) as well as GFAP expression in the Mu¨ller cell population and
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Fig. 1. Retinal detachment in the experiments used physiological weightlessness simulated by clinorotation. (A) - view of the newt’s eye after retinal detachment. Bar: 500 lm; (B) –Semithin section (1 l) of individual Mu¨ller glial cell with [3H]-TdR labeled nuclei (arrow).
Fig. 2. Increase of cell population of Mu¨ller glia in the retina of clinorotated animals as compared with controls at the 16th day after retinal detachment and 7th day of rotation. (A) - Absolute number of [3H]-TdR labeled cells in the central part of the retina in rotated (dark column) and control (light column) animals. ONL - retinal outer nuclear layer, INL – inner nuclear layer, GCL - ganglion cell layer. (B) - Relative number (per one section, M ± m) of Mu¨ller glial cell processes in different zones of the retina in rotated (dark column) and control (light column) animals. Dors – dorsal, Centr – central, Ventr – ventral retinal regions. An increase of a number of [3H]-TdR labeled cells in INL (area of Mu¨ller cell bodies’ location) correlates with the increase of a number of Mu¨ller glial cell processes at the peripheral and central retina.
FGF2 and HSP90 expression in the eyes of space-flown and synchronous control newts. Using monoclonal antibodies, FGF2 expression playing the key role in the eye development and regeneration was examined by immunohistochemical methods. Sections from all animal groups (basal control, flight, synchronous control) were treated simultaneously following the same protocol, and images were obtained after the same exposure time. In basal controls that were at stage II of lens regeneration, immunospecific fluorescence was detected in the retinal interior and in the growth zone. In synchronous controls, that were at stage VII, and in the flight animals, that were at stage VIII, growth zone cells were also stained, some cells showing more intense staining at their periphery. Both flight and synchronous animals showed fluorescence in the iris vascular membrane, cornea epithelium and eye growth zone. However, the intensity of FGF2 expression in the flight animals was higher than in the controls (Fig. 3A and B). Using immunochemistry methods, we also investigated the expression of HSP90 heat shock protein as another potential regulator of eye regeneration in space. We found for the first time that antibodies against the protein were most actively bound to retinal Mu¨ller cell. In basal controls, the reaction, although weak, was sufficient to identify immunofluorescence localization. We detected it in the perikaria and accessory prolongations of glial cells, as well
as in the photoreceptor layer and in the retina, where it was less visible. On the whole, the staining intensity (or fluorescence level) was greater in the sections of regenerating eyes of the flight newts compared to that in basal and synchronous controls. The fluorescence level was the highest at the site of expanding end-feet of accessory prolongations of glial cell (Fig. 4A and B). Moreover, Mu¨ller cell processes in the outer retina of the flight animals also showed a distinct immune reaction. The staining intensity of glial cells of the regenerating eyes of synchronous controls was significantly lower, reaching the level between that in basal controls and in flight animals. In Foton-M3 experiment, GFAP expression in retinas of space-flown, ground control (kept at 1 g), and basal control newts was quantified using microscopy, immunohistochemistry, and digital image analysis. It was found that Mu¨ller cell processes of nonoperated animals displayed relatively low GFAP immunolabeling. A low level of immunoreactivity was also observed in basal controls. In contrast, retinas of spaceflown animals showed greater GFAP immunoreactivity associated with both an increased cell number and a higher density of intermediate filaments. Using custom designed software, we measured means of the stained area (in pixels) as a percent of the entire field (in pixels). Random fields of the same size were marked in the images of central sites of eye retinas isolated from the newts of all three groups and
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Fig. 3. Expression of fibroblast growth factor (FGFb) in the eye vascular network (arrows) of “flown” (A) and synchron (B) newts studied soon after landing of Foton M-3. Bar: 20 lm. In the presented case immuno-fluorescence intensity evaluated by means of estimation of equally stained areas (tolerance = 40) was: A – 3167 and B – 5321 pixels.
Fig. 4. Expression of heat shock protein 90 (HSP 90) in the retinal Mu¨ller cell endfeet (arrows) of retinal Mu¨ller cells in flight (A) and synchron (B) animals. GL – ganglion cell layer, IPL – inner plexiform layer, INL – inner nuclear layer of the retina. Bar: 20 lm.
group of intact animals. As can be seen in Fig. 5, the parameter was greater in the flight animals when compared to the controls. 4. Discussion The results of newt experiments onboard Bion-11 and Foton-M3 as well as in clinorotation demonstrated that the gliotic response of the retinal macroglial population was stimulated by an altered gravitational field. During clinorotation the proliferation of Mu¨ller cells and the number of their accessory prolongations in the detached retina increased when compared to the controls. This phenomenon manifested a response of the macroglial population to the shift in retinal physiology initially caused by the ret-
Fig. 5. Computer data of relative intensity of GFAP expression in the square unit in the central area of the retina of intact newts (black), basal control (white), synchronous control (light grey) and flight (dark grey column) animals.
ina detachment and subsequently enhanced by clinorotation. With respect to the parameters we measured, the most significant differences were detected in the central retina. It is important to note here that this is the area where serious eye disorders, e.g., macular degeneration, develop (Nowak, 2006). The Bion-11 experiment, in which the retina was regenerating after the optic nerve was cut, revealed changes in the glial population, viz., GFAP upregulation assumed to be an indicator of retinal stress. For instance, in response to the retinal detachment or bright light damage the concentrations of GFAP, Mu¨ller cells and their prolongations increase. This type of reaction is observed in response to different retinal injuries and in various animal classes. Study of GFAP expression in the retina of newts, an animal model used in space-flown regeneration experiments, may provide insight into the role the Mu¨ller glia plays in an altered gravitational environment. Examination of the Mu¨ller cell population in the FotonM3 experiment gave support to the observation concerning glial response enhancement in the flight newts compared to the controls. Similarly to what was found in previous flights, GFAP expression was up-regulated when measured by immunospecific fluorescence intensity. However, in the Foton-M3 newts GFAP up-regulation developed together with FGF2 and HSP90 up-regulation. It was previously demonstrated that FGF2 was involved in the induction and regulation of cell proliferation as well as in the expres-
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sion of proteins of intermediate filaments of Mu¨ller cells (Lewis et al., 1992). Moreover, it was suggested that the phenotype changes of Mu¨ller cells could be induced by exogenous growth factors in the absence of retinal damage (Fisher et al., 2004). From our perspective, it is important to consider potential interactions of FGF2 and HSPs as well as HSPs and GFAP. The HSP90 presence in the regenerating eye retina and particularly in Mu¨ller cells suggests that this protein can be engaged in the Mu¨ller cell gliotic response. HSP expression in the same cell population in response to a stress was demonstrated earlier both in vivo (Gohdo et al., 2001) and in vitro (Hauck et al., 2003). We believe that an enhancement of the Mu¨ller cell gliotic response we detected in three different experiments using different methods of retinal injury was not induced by gravity changes per se but was mediated by changes in the whole organism. There are observations indicating intraocular pressure (IOP) changes in humans exposed to simulated microgravity (Kuzmin, 1984), real space missions (Schwartz et al., 1993) as well as to rapidly changing g levels (Kroll et al., 1987). However, rats subjected to laser-induced IOP elevation showed loss of some ganglion cells which was followed by a gradual and sustained increase in GFAP immunoreactivity (Lam et al., 2003). Another cause of up-regulated gliotic response to the microgravity environment may be ion balance changes in the eye vitreous body. As known, variations in the internal environment (including ion composition) of the retinal radial glial cells can modify their function and the spectrum of molecules they synthesize (Steinbach and Schlosshauer, 2000). On the whole, the phenomenon is aimed at retina protection. In conclusion, adequate understanding of the signaling mechanisms implicated in gliotic alterations of Mu¨ller cells in the space environment is essential for preventing excessive development and destructive effects of the glia in the retina. References Anton, H.J., Grigoryan, E.N., Mitashov, V.I. Influence of longitudinal whole animal clinorotation on lens, tail, and limb regeneration in Urodeles. Adv. Space Res. 17, 55–65, 1996. Dyer, M.A., Cepko, C.L. Control of Mu¨ller glial cell proliferation and activation following injury. Nat. Neurosci. 3, 873–880, 2000. Erickson, P.A., Fisher, S.K., Gue´rin, C.J., Anderson, D.H., Kaska, D.D. Glial fibrillary acidic protein increases in Mu¨ller cells after retinal detachment. Exp. Eye Res. 44, 37–48, 1987. Fisher, A.J., Omar, G., Eubanks, J., McGuire, Ch.R., Dierks, B.D., Reh, T.A. Different aspects of gliosis in retinal Mu¨ller glia can be induced by CNTF, insulin, and FGF2 in the absence of damage. Mol. Vis. 10, 973–986, 2004. Garcı´a, M., Vecino, E. Role of Mu¨ller glia in neuroprotection and regeneration in the retina. Histol. Histopathol. 18, 1205–1218, 2003. Gohdo, T., Ueda, H., Ohno, S., Iijima, H., Tsukahara, S. Heat shock protein 70 expression increased in rabbit Mu¨ller cells in the ischemiareperfusion model. Ophthalm. Res. 33, 298–302, 2001. Grigoryan, E.N. Alternative intrinsic cell sources for neural retina regeneration in adult urodelean amphibians, in: Chiba, Ch. (Ed.), Strategies for Retinal Tissue Repair and Regeneration in Vertebrates:
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