- Email: [email protected]
Microgravity effects on neural retina regeneration in the newt
Adv. Space Res. Vol. 22, No. 2, pp. 293-301, 1998 01998 COSPAR. Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain 0273-I 177/98$19.00 + 0.00 PII: SO273-1177(97)00050-7
Pergamon
MICROGRAVITY EFFECTS ON NEURAL RETINA REGENERATION IN THE NEWT E. N. Grigoryan*, H. J. Anton**, and V. I. Mitashov’ *Instituteof Developmental Biology, Russian Academy of Sciences, 26 Vanilov str., Moscow 117808, Russia **Zoological Institute, Universiq of Cologne, 119 Weyertal str., Cologne D 50923, German)
ABSTRACT Data on forelimb and eye lens regenerationin in urodeles under spaceflight conditions (SFC) have been obtained in our previous studies. Today, evidence is available that SFC stimulate regeneration in experimental animals rather than inhibit it. The results of control on-ground experiments with simulated microgravity suggest that the stimulatory effect of SFC is due largely to weightlessness. An original experimental model is proposed, which is convenient for comprehensively analyzing neural regeneration under SFC. The initial results described here concern regeneration of neural retina in Pleurodeles waltl newts exposed to microgravity simulated in radial clinostat. After clinorotation for seven days (until postoperation day 16), a positive effect of altered gravity on structural restoration of detached neural retina was confirmed by a number of criteria. Specifically, an increased number of Mtillerian glial cells, an increased relative volume of the plexiform layers, reduced cell death, advanced redifferentiation of retinal pigment epithelium, and extended areas of neural retina reattachment were detected in experimental newts. Moreover, cell proliferation in the inner nuclear layer of neural retina increased as compared with control. Thus, low gravity appears to intensify natural cytological and molecular mechanisms of neural retina regeneration in lower vertebrates. 01998 COSPAR. Published by Elsevier Science Ltd. All rights reserved
INTRODUCTION In our previous studies, we showed that space flight conditions stimulate cell proliferation and eyes lens and limb regeneration in newts (Mitashov et al., 1987; 1990; 1996; Grigoryan et al., 1992). In recent experiments, we found that the rates of cell proliferation and lens, tails and limb regeneration increase also under conditions of microgravity simulated by clinorotation (Anton and Grigoryan, 19934 b; Anton et al., 1996). Urodeles demonstrates a unique ability to regenerate the retina damaged in different ways, including its complete and partial removal, detachment, or cutting of the optic nerve (Grigoryan, 1996). Retina detachment in the newt is an interesting model for analyzing effects of microgravity on neural regeneration in vertebrates. This operation leads to cell proliferation, transformation, and migration both in neural retina @JR)and underlying retinal pigment epithelium (NRP), which provide for neuronal retina restoration (Grigoryan et al., 1996) and, in some cases, for the formation of additional retinal structures (Grigoryanand Mitashov, 1985;Grigoryan, 1992). When developing under conditions of microgravity, these 293
294
E. N. Grigotyan, et al
processes should have certain peculiarities, as it was demonstrated for limb and eye lens regeneration in newts. On the other hand, data on the effect of exposure to microgravity on retina and optic nerve regeneration in vertebrates are virtually absent, although this problem, as well as the general problem concerning the pattern of regeneration processes in vertebrates under spaceflight conditions, has both theoretical and practical significance. Studies on the state of human visual system revealed changes in retinal blood circulation and intraocular pressure occurring under conditions of simulated microgravity (Kuz’min, 1984) and rapidly changing g-loads (Kroll et al., 1987). These microgravitydriven changes may have significance for restoration of the vertebrate eye after neural retina detachment. Hence, studies on the processes occurring under conditions of changing gravity in vertebrate retina restoring after its detachment are quite interesting and important. In this work, we tried to reveal specific morphological features and types of regenerative cell response in the neural retina regenerating after its complete detachment in newts exposed to microgravity simulated by clinorotation.
MATERIALS AND METHODS Newts PZeurode1e.swaiti from the aquarial room of the Institute of Developmental Biology, RAS were used in experiments. The animals were selected by size (100-105 mm) and weight (5.5-6.0 g). Neural retina has been detached in both eyes of the newts nine days before the clinorotation as follows. The lens was first removed from the animals narcotized in physiological saline for amphibia (0,65% NaCl) containing MS-222 (1:3000) through the incision of cornea in the pupil area. Then a slight atraumatic strain was created by slightly pressing of the limb circle by glass rod with a round tip until a moment when folds of detached retina became visible through the pupil. Two animals were injected intraperitoneally with [3HYJ-thymidineat 5 mCi/g (specific activity 4,6 Ci/mM, “Isotope”, Moscow, Russia) nine days after operation (basal or conventional control) and 3 h before fixation. HaIf of the remaining newts were mounted in the clinostat. The other half served as a control (see below). The so-u&d “radial” (radially - oriented) fast rotating clinostat was custom-built in the workshops of the Zoological Institute, University of Cologne, Germany. It consisted of six disks driven by an electric motor (Figure 1). The rotation axis passed through the centers of the disks. Constant lOO?! humidity required by newts was maintained by submerging of disks once per revolution into water that filled l/3 of the clinostat chamber. The animals were fixed on the clinostat disks using elastic cotton net (Hartmann, Germany) in the way that the rotation axis passed aposteriorily through the frontal part of the head where the eyes were located. Insignificant changes in body position were allowed to reduce stress in the animals. Maximal deviation of the newt eyes from the rotation axis was no more than 8 mm, and this caused some gravity vector changes. Our calculations made according to Silver (1976), showed that, at 60 rpm, gravity force varied from 8,05 x lo‘3 to 3,21 x lo’* g. Clinorotation at 60 rpm was performed in the dark at 21-22” C for seven days. Equal number of the operated animals (4 newts) served as a control and were kept under similar Fig. 1. Devices used in the study on conditions in an aquarium, on flter paper saturated with fimctional microgravity influenceupon water. Injections of [31-I+thymidineto animals of both groups neural retina regeneration in the newt were made immediately a&r switching off the clinostat and
Microgravity
295
Effects on Neural Retina Regeneration in the Newt
three hours before fixation. Cell proliferation and morphological changes in different zones of detached retina were studied using autoradiographic, electron microscopic and histological analysis followed by morphometry (for a detailed description of methods see: Mitashov et al, 1987; Grigoryan et al., 1996). Morphometric measurements were made with the aid of ocular micrometer in sagittal sections (5 mkm) stained with nuclear fast red. The state of retina after its complete detachment in the control and experimental newts was evaluated using such criteria as the number of proliferating and degenerating cells, absolute thickness of the retina and relative thickness of its layers, the number of Mtiller cell processes, the number of macrophages, development of the optic nerve, reaction of the retinal pigment epithelium, etc. (Table 1). Measurements were made in the every second or&ird section in a complete series of eye cross-sections representing the central zone of the retina (equivalent to the size of the pupil). Data of measurements and calculations were processed statistically.
Table 1. Criterion chosen for study on simulated microgravity influence upon neural retina regeneration after its detachment in the newt Pleuroakles Walt1 Criterion “Negative” l.Celldeathin NR 2. RPE cell responses (transformation, proliferation and migration) 3. Phagocytic cell reaction “Positive” 1, Development of photoreceptor cell outer segments 2. Thickness of nuclear layers 3. Thickness of plexiform layers 4. Optic nerve development 5. Extension of NR-RPE reattachment 6. Volume of vitread endfeet ofMtiller glial cells 7. Cell proliferation in inner nuclear layer
Experiment
Control
+ + +
++ ++ ++
+t i+ +t
+ + +
U
+
U
+
U
+
U
+
RESULTS Morphological analysis of neural retina atIer its complete detachment in newts demonstmted firstly that the operation procedure was relatively autraumatic. No breaks or cuts in retina and retinal pigment epithelium were observed in eyes of the operated newts during the observation period: neural retina was detached as a whole, and retained partly destructed photoreceptor outer segments. After detachment, the retina survived for a long time: in operated animals that were additionally fixed one month after operation, no signs of retina degeneration were observed. Development of a characteristic responses of eye tissues to the lens removal and retina detachment was observed on the 9th day after the operation (basal or conventional control). Cells of retinal pigment epithelium and iris dediBerentiated and begun to proliferate. A number of [3H]-Tdr-labeled cells in these areas constituted 8 to 12%. An increase in macrophage number was observed near the pigment epithelium, dorsal iris, and in the local areas of cell death, usually deep inside of retinal folds. Some of the
2%
E. N. Grigoryan, et al.
mactophages were labeled. A small number of [jH]-Tdr-labeled cells was also observed in iris and cornea. This also indicates the beginning of post-operation eye regeneration. Lens regeneration was at the I-II st. according to accepted classification of Yamada (1967). The content of labeled cells was quite low in the retina which was thinned and located in the eye cavity forming folds at this time.The labeled cells were localized in the inner nuclear layer (ML) , and very rarely in other retinal nuclear layers (ONL, CCL) and in the area of the optic nerve outlet. Absolute numbers of the labeled cells and their diitribution among different retinal areas after mounting the animals into the clinostat are shown by Figure 2. The following changes in the morphological state and cell proliferative activity of the detached retina have been observed at the 16th post-operation day and after the end of chnorotation and in the ground control. The total number of proliferating retinal cells was lower in both groups of animals than at the 9th postoperation day (basal control). This 2-3 times decrease was similar in all the analyzed areas (Figure 2). Both on the 9th and 16th days post- operation the majority of r3HJ-Tdr labeled cells were associated with inner nuclear layer of the central part of the detached 220 retina. Additionally applied electron microscopy 200 analysis and analysis of semi-thin sections have 180 shown that the population of cells incorporated 180 13Hj-Tdr in inner nuclear layer is represented by I~ Mtillerian glial cells and by interneurons, 120 presumably bipolats. In this case the labeled nuclei 100 were located deep within this layer and had a radial 80 orientation. We could not detect very signifkant 6o differences in proliferative activity of retinal cells in *O ONL INL GCL the control animals and in the animals subjected to n=lOO;N=S clinorotation because of a low general proliferation q CENlR(C) q CENTRQ Doa (C) m oCf=(E) [ fl level in both cases. One can only note that there were more proliferating cells in the central zone of Fig. 2. Absolute number of [3H] -Tdr labeled the retina, and in particular in the inner nuclear cells in dorsal and central parts of detached neural layer of the clinostat -exposed animals (Figure 2). retina a&r chnorotation and in the control Partial cell death occurs in avascular newt retina a&er its complete detachment. It is due to disorders on cell trophy which is carried out under normal conditions at the expense of choroidal circulation. An absolute number of necrotic cells in the detached retina has been counted in 100 central sections on the 9th day _ n=loo;N=l and then on the 16th day post-operation in the SO0 experiment (7th day of chnorotation) and in the control. As follows from the Figure 3, the number 600 of necrotic cells is rather high on the 9th day post400 operation (particularly in the dorsal area of the retina), but it decreases sharply by the 16th day. 200 The most sign&ant decrease in the number of n v-necrotic c4Als occurs in the clinostat-exposed 16VWO* 9.0" animals as compared to the control (Figure 3). l-daytiopemtkw--dayaftwrotation Thickness of the nuclear layers and the whole retina between inner and outer limiting membranes Pig. 3. Absolute number of necrotic cells obaerved 1 different areas of detached neural retina before (excluding the outer segments of the photoreceptor m cells) served as an another criterion of the and a&r clinorotation and in the control morphological state of the detached retina. The total thickness of the detached retina was increased on the 16th day post-operation as compared to the 9th
Microgravity Effects on Neural Retina Regenerationin the Newt
297
day (Table 2). The increase in thickness is due to limited coordinated growth of its nuclear layers where proliferation was detected (Table 2) and to reconstitution of the cytoplasm volume. No preferential growth of a retinal zone was observed. No sign&ant difference in this parameter was observed in the retina of the clinostat-exposed animals as compared to the control ones. At the same time measurement of the total thickness of the morphologically nuclear&e plexiform layers (calculated by subtraction of the total thickness of the nuclear layers from the total thickness of the retina) rev&s their significant increase upon clinorotation at least in the dorsal and central areas of the neural retina (Table 2). Table 2. Total thickness of the detached retina (NR), thickness of its nuclear layers, and relative thickness of plexiform layers (PL) before and after the clinirotation and in the control. Days
9" 0"" 16" 0"" 16" 7'"
92,1*1.9 93.7*1,9 104,2+2,3
28,Ok I ,0 27,l zO,8 28,1*1,1
Dorsal area 35,421,3 3452 1.2 39,3? 1,l
10,4&,5 12,4&,6 11,5&1,4
19.9% 21,1% 24.3%
lO,l?xl,3 13,1&0,5 12,4*,3 GCL
16,6% 25,3% 36.2% PL
centnd area
60,7-c1,4 21.6rO.6 9" 0"' 27,9~1,2 100,6*2,5 16'0"" 27,8ti,9 112,4%2,2 16" 7"" ONL NR -*-days after operation, -**days after rotation.
19,34,7 34,2* 1,o 31.6ti.9 INL
We have also counted a number of long radial processes (accessory prolongations) of the M&&n glial cells in the inner plexiform layer in different areas of the retina. These processes reach towards the vitreous body through the inner part of the retina and can be visualized using NS-9 and ZS-I filters @ST 941 l60). Counting of fibers of these cells in different lP=QN=d n=loop6 F#n;N=8 areas of the retina demonstrates that their number 45 increases by the 16th day post-operation, and that 40 this increase is significantly higher in the clinostat36 exposed animals (Figure 4). Interestingly, a greater 30 increase of the number of processes isobservedin 25 20 the central area of the detached neural retina. We have also analyzed other eye tissues besides the Is above-described studies of changes in the detached lo retina. The changes in morphology of these tissues 5 9v *- daytier cqet~~~ - day alter ;%n may also reflect the effect of clinorotation on ( El DORS q CENTR 61 VENTR ] regeneration processes in the eye. Eye lens regenerates after its removal during the operation. Fig. 4. Number of Miiller glial cell processes in It is formed on the 16th day post-operation from different areas of detached neural retina before and the cells of the distal part of dorsal iris. The after clinorotation and in the control regeneration rate was somewhat higher in the ciinostat-exposed animals than in the control: III-IV vs. II-IV stages respectively. Optic nerve which is retained during the operation was better developed afbx the clinorotation as determined by its diameter in the area of ocular nodule and its morphology in the area of its going out of eyeball. Retinal pigment epithelium undergoes significant changes directed to regeneration of the retina after its detachment In the present work we have chosen time intervals where we could only observe the beginning of this process: cell dedifF&ntiation, cell proliferation and cell transformation into phagocytes as was described earlier (K&e, 1973). These processes were observed both in the control animals and in the
298
E. N. Grigoryan, et al.
clinostat-exposed ones. The differences were found in the extension rate of the pigment epithelium areas exhibiting this response to the retinal detachment. The retinal pigment epithelium areas expressing the morphological changes were much more reduced in the experimental animals as compared to the control. Inversely, the retinal pigment epithelium redifferentiation zones were larger in the clinostat-exposed animals. Cells of redifferentiated retinal pigment epithelium were in close reapposition to photoreceptor outer segments of morphologically normal retina. At the same time we did not measure the size of reattachment regions because of a possibility of artefacts in the morphological pattern of interaction of the retina and pigment epithelium due to relatively harsh fixation conditions. We do not supply data on changes in morphology of photoreceptor outer segments because of the same reason. We have counted the number of macrophages in the eye cavity near the detached retina and within its layers before and after clinorotation, and in the controls (9th and 16th day post-operation, respectively). A significant number of macrophages were observed before the clinorotation (50-300, SO-300 and 50-150 in the dorsal, central, and ventral areas of one eye, respectively). The number of macrophages decreased right afler the clinorotation and constitutes 5-50, 5-60, and 5-40 in the same zones, respectively. At the same time a number of macrophages was somewhat higher in the eye dorsal and central zones of the control animals: 5-150, and 5-200, while in the ventral zone it was similar to that of the experimental animals (530). DISCUSSION Our data suggest that exposure to microgravity simulated by clinorotation at 60 rpm may cause a number of changes in the morphological state of retina after its detachment. According to the chosen criteria (Table 1), these changes concern largely the development of the plexiform layers of the detached retina, Mtiller glial cells, and, to a lesser extent, the number of macrophages, proliferating and degenerating retinal cells, and the ability of the retina to restore apposition to RPE. They occurred mainly in the dorsal and central zones of the detached retina. We found that an increase in thickness of the retina, resulting from growth of its reticular layers, in newts subjected to clinorotation for seven days is greater than in control newts kept in an aquarium. The plexiform layers appear to grow due to an increase in the number and length of neuronal and glial cell processes, which form these layers. Mtiller glial cells, located radially in the inner nuclear layer and representing the major glial component of the retina, belong to this growing population of cells. Published data indicate that these cells play multiple roles in the retina. They can easily change their phenotype and undergo biochemical changes depending on the state of retinal neurons and their relationships (Moscona, 1987). Mtiller cells can synthesize various macromolecules supporting neuron functioning and maintaining extracellular microenvironment during prolonged retinal detachment (Lewis et al., 1989). These nonneuronal cells are a structural component of the adult retina and play a morphogenetic role in the developing retina (Wolburg et al., 1991). Finally, they supposedly play the role of macrophages, being the major retina “cleaners” (Nishizono et al., 1993), and are responsible for myelin production in the fibrillar layer of the, optic nerve (Huges and La Velle, 1974). All this suggests that Mtiller cells maintain the retinal homeostasis. Published data and our results show that all the above functions become activated after retinal detachment. Morphologically, it is manifested in cell proliferation, hypertrophy, migration, and nuclear translocations (Guerin et al., 1990; Grigoryan et al., 1996). These processes are a typical cell response to changing environment and may be activated by factors other than retinal detachment. In our experiments, the loss of topological and functional relationships between the retina and RPE was accompanied by changes in gravity caused by clinorotation. Hence, the regenerative response of Miiller glial cells to retina detachment, manifested in an increase in the number of accessory processes, was enhanced in this case by altered gravity. These data agree with the results of our study on cell proliferation in detached retina: although the level of proliferation in the latter was generally low, newts subjected to clinorotation demonstrated an increase in proliferative activity of cells of the inner nuclear layer in the central zone of
Microgravity
Effects on Neural Retina Regeneration in the Newt
299
the retina (Figure 2). Data on radial orientation of [3H]-TdR-labeled nuclei and morphology of labeled cells in ultra- and semithin sections of the retina, which resembled that described by Miiller (1862) and Uga and Smelser (1973), suggest that Mtiller glial cells make a significant contribution to the pool of proliferating cells in the retinal inner layer. These cells were activated for proliferation first by retinal detachment and then by clinorotation. Hence, clinorotation-simulated microgravity may activate numerous natural functions and change the behavior of retinal glial cells after retinal detachment, and this cell response leads to more successful structural and functional regeneration of the neural retina. We found that simulated microgravity affects other parameters of retina regeneration. According to our data, the number of retinal cells degenerating after detachment in newts subjected to clinorotation is much less than in control animals. The same concerns the number of macrophages in the retina and around it. Macrophages appear in the eye cavity in response to eye damage and reflect a long-term (in poikilotherms) inflammatory reaction (Mitashov et al., 1979). Disappearance of macrophages indicates cessation of this reaction and the absence of additional signals for macrophage migration into the eye cavity. According to this criterion, simulated microgravity appears to have a favorable effect on regeneration of the detached retina. Better development of the optic nerve, a higher rate of lens regeneration, and behavior of RPE cells in experimental animals provide additional evidence for this conclusion. Most of the above-mentioned differences in the state of detached retina in animals subjected to clinorotation, activation of Muller cell growth in particular, were better manifested in the dorsal and central areas of detached retina rather than in its ventral part. The adaptive differences in growth capacity of Muller cells between the central and peripheral zones of the retina were described previously and attributed to the fact that thickness of the retina decreases toward the eye growth zone (P&a et al., 1989). However, our previous experiments on eye lens regeneration under microgravity simulated by clinorotation (Anton and Grigoryan, 1993a) also demonstrated preferential activation of proliferation in tissues of the dorsal eye area (eyelid skin, iris, and cornea) These spatial differences are attributed either to intrinsic peculiarities of cells located in different zones of a certain tissue or to peculiarities of their interactions with surrounding cells and tissues. The latter explanation seems likely. Although the stimulatory effect of microgravity on lens and limb regeneration in newts was first demonstrated in 1985 (Mitashov et al., 1987) and has been studied since then from different aspects under the actual spaceflight conditions (Grigoryan et al., 1992, Mitashov et al., 1990, 1996) and in experiments with simulated microgravity (Anton and Grigoryan, 1993a,b; Anton et al., 1996), mechanisms responsible for the effect of microgravity on regeneration processes remain unclear. In the case of retina regeneration, microgravity may affect the expression of known growth-stimulating factors, their circulation in eye cavity or transport to target ceils. For example, recent experiments revealed tissuespecific changes in the level of TGF-b mRNA in skeletal tissues of rats growing under spaceflight conditions (Westeriind and Turner, 1995). Microgravity can also specifically alter cell adhesion in the retina, whose role in triggering retina regeneration is currently being investigated (Raymond et al., 1995). Moreover, studies on urodeles exposed to spaceflight conditions revealed profound changes in their calcium metabolism (see: Berezovska et al., this volume), and calcium ions are known to play an important role in the transduction of hormonal and neuronal signals. This suggests the involvement of microgravity-induced calcium changes in stimulating regeneration of neural retina in the newt. A general increase in the level of retinal cell metabolism resulting from an adaptive reaction (generalized stress) of the whole organism to altered gravity may also stimulate this process. These ideas require further experimental confirmation. In subsequent experiments, we plan to use other models of neural retina regeneration in newts (in particular, regeneration after cutting the optic nerve and blood vessels) for analyzing the role of effects caused by microgravity in neural regeneration in space.
300
E. N. Grigoryan, er ai.
ACKNOWLEDGMENTS We are grateful to V. A. Poplinskaya for her technical assistance in processing experimental material. This work was supported in part by DFG and by contract NAS 15-10110. REFERENCES Anton, H. J. and E. N. Grigoryan, Simulated Microgravity Induces Increase of Regeneration Rate, Cell Proliferative Activity, and Enhancement of Size of Regenerate during Wolffran Lens Regeneration in the Newt. 86. ye&. Deutsche Zooi. Ges. 1993. Salzburg (Kurzpublikationen), 86,204 (1993a). Anton, H. J. and E. N. Grigoryan, Altered Influence of Gravity Can Provide Long-term Effect on Forelimb and Tail Regeneration in the Newt. (Preliminary Results of Experiments on Simulated Microgravity), 86 Vehr. Deutsche Zool. Ges. 1993. Salzburg (Kurzpublikationen), 86,203 (1993b). Anton, H. J., E. N. Grigoryan and V. I. Mitashov, Influence of Longitudinal Whole Animal Clinorotation on Lens, Tail, and Limb Regeneration in Urodeles, A& Space Res., 17, (6/7) 55 (1996). Berezovska, 0. P., N. V. Rodionova, E. N. Grigoryan et al., Effect of Microgravity on Activity of Osteoclasts in Newts, Adv. Space Res., (in press). Grigoryan, E. N. Eye Regeneration after Retina Detachment and Tear-off in the Newt Pleurodeles waltl, Bulletin of Russian Acad Sci., ser. Biol., N 1, 18 (1992). Grigoryan E. N., Retina of Urodeles as a Model for Study on Retinal Regeneration Capacities in Other Vertebrates, Ontogenez, 27, N 3, 173 (1996). Grigoryan E. N. and V. I. Mitashov, Cultivation of Pigment Epithelium in a Cavity of Lens-ectomized Newt Eye, Ontogenez, 16, N 1,34 (1985). Grigoryan, E. N., I. P. Ivanova and V. A. Poplinskaya, Identification of a Novel, Intrinsic Cell Source for Neural Retina Regeneration after its Detachment in Newts. I. Morphological and Quantitative Studies. Bulletin of Russian Acao! Sci., ser. Biol, , N 3, 3 19 (1996). Grigoryan, E. N., E. A. Oigenblick, S. Y. Tuchlcova et al., The Influence of Space Flight Factors on Lens and Limb Regeneration in Newts, in Results of Investigations aboard Russian Biosatellites, pp. 345350, Nauka, Moscow (in Russian) (1992). Guerin, Ch. J., D. H. Anderson and S. K. Fisher, Changes in Intermediate Filament Immunolabeling Occur in Response to Retinal Detachment and Reattachment in Primates, Invest. OphthaZ. Vis. Sci., 31, N 8, 1474 (1990). Hughes, W. F. and A. La Velle, On the Synaptogenic Sequence in the Chick Retina, Anat. Rec., 179,297 (1974). Keefe, J. R., An Analysis of Urodelian Retinal Regeneration: III. Degradation of Extruded Melanin Granules in Notophthalmus viridescens, J. Exp. Zoof., 184,233 (1973). Kroll, G., J. Draeger, H. Brande, et al., Untersuchungen uber den Intraocularen Druck bei rasch wechselnden g-Belastungen in der Z-Achse, Mitt. Dtsch. Ges. Luft-und Raumfahrtmed, 2, 10 (1987).
Microgravity Effects on Neural Retina Regeneration in the Newt
301
Kuz’min, M. P., Blood Circulation in the Human Eye Retina and Eye Pressure in Conditions of Simulated Zero Gravity, in Space Anthropology Techniques andMetho& of Stuc&, p.447. Nauka, Leningrad, (in Russian) (1984). Lewis, G. P., P. A. Erickson, C. J. Guerin, et al., Changes in the Expression of Specific Mueller Cell Proteins during Long Term Retinal Detachment, Exp. Eye. Res., 48,93 (1989). Mitashov, V. I., V. I. Starostin, A. I. Sludskaya, et al., An Autoradiography Study of Macrophagal Activity during Eye Regeneration in Adult Newts, Ontogenez, 10, N 4,365 (1979). Mitashov, V. I., E. N. Grigoryan, S. Y. Tuchkova et al., Organs and Tissue Regeneration in Amphibia under the Space Flight Conditions, in Life Science Research in Space. pp. 299-303, ESA and ESTEC Publ. Division, Netherlands (1987). Mitashov, V. I., E .N. Grigoryan, S. Y. Tuchkova, et al., Lens and Limb Regeneration in the Newt during and a&r 13 Day Long Space Flight, in Microgravity is a Tool of Developmental Biology, ESA and ESTEC Publ. Division, Netherlands, 85 (1990) . Mitashov, V. I., N. V. Brushlinskaya, E. N. Grigoryan et al., Regeneration of Organs and Tissues in Lower Vertebrates during and after Space Flight, A&. Space Rex, 17, (6/7) 241 (1996). Moscona, A. A, Role of Cell Contact in Phenotype Stability and Modification: Studies on Retina Cells, Amer. Zool., 27, 137 (1987). Mtiller, H., ijber das Auge des Chamiileons mit vergleichenden Bemerkungen, Zietschz?., 3, 10 (1862).
Wiirzb. Naturw.
Nishizono, H., Y. Murata, M. Tanaka et al., Evidence that Mueller Cells Can Phagocytize Egg-lecitincoated Silicone Particles, Tissue and Cell, 25, 305 (1993). Prada, F. A., M. M. Magalhaes, A. Coimbra et al., Morphological Differentiation of the Mueller Cell: Golgi and Electron Microscopy Study in the Chick Retina, J. Morphoi., 201, 11 (1989). Raymond P.A, J.E. Bra&d, J. K. Knight et al., Role of Cell-cell Interactions in the Regulation of Retinal Regeneration, in Publ. of 6th Internat. Sjmp. on Neural regeneration, Asilomar, USA, 29 (1995). Silver, I. L. The Dynamics of a Discrete Geotropic Sensor Subject to Rotation-induced Gravity Compensation, J. Z’heor.Biol., 61,353 (1976). Uga, S., and G. K. Smelser, Comparative Study of the Fine Structure of Retinal Mueller Cells in Various Vertebrates, Invest. Ophthalmol., 12,434 (1973). Westerlind, K. C. and R. T. Turner, The Skeletal Effects of Spaceflight in Growing Rats: Tissue-specific Alterations in mRNA Levels for TGF-beta, J. Bone andMineral Rex, 10,843 (1995). Wolburg, H., E. Willbold, and P. G. Layer, Muller Glia Endfeet, a Basal Lamina and the Polarity of Retinal Layers Form Properly in vitro only in the Presence of Marginal Pigment Epithelium, CeN Tisnre Res., 264,437 (1991). Yamada, T., Cellular and Subcellular Events in Wolffian Lens Regeneration, Curr. Top. Develop. Biol., 2,247 (1967).
Pergamon
MICROGRAVITY EFFECTS ON NEURAL RETINA REGENERATION IN THE NEWT E. N. Grigoryan*, H. J. Anton**, and V. I. Mitashov’ *Instituteof Developmental Biology, Russian Academy of Sciences, 26 Vanilov str., Moscow 117808, Russia **Zoological Institute, Universiq of Cologne, 119 Weyertal str., Cologne D 50923, German)
ABSTRACT Data on forelimb and eye lens regenerationin in urodeles under spaceflight conditions (SFC) have been obtained in our previous studies. Today, evidence is available that SFC stimulate regeneration in experimental animals rather than inhibit it. The results of control on-ground experiments with simulated microgravity suggest that the stimulatory effect of SFC is due largely to weightlessness. An original experimental model is proposed, which is convenient for comprehensively analyzing neural regeneration under SFC. The initial results described here concern regeneration of neural retina in Pleurodeles waltl newts exposed to microgravity simulated in radial clinostat. After clinorotation for seven days (until postoperation day 16), a positive effect of altered gravity on structural restoration of detached neural retina was confirmed by a number of criteria. Specifically, an increased number of Mtillerian glial cells, an increased relative volume of the plexiform layers, reduced cell death, advanced redifferentiation of retinal pigment epithelium, and extended areas of neural retina reattachment were detected in experimental newts. Moreover, cell proliferation in the inner nuclear layer of neural retina increased as compared with control. Thus, low gravity appears to intensify natural cytological and molecular mechanisms of neural retina regeneration in lower vertebrates. 01998 COSPAR. Published by Elsevier Science Ltd. All rights reserved
INTRODUCTION In our previous studies, we showed that space flight conditions stimulate cell proliferation and eyes lens and limb regeneration in newts (Mitashov et al., 1987; 1990; 1996; Grigoryan et al., 1992). In recent experiments, we found that the rates of cell proliferation and lens, tails and limb regeneration increase also under conditions of microgravity simulated by clinorotation (Anton and Grigoryan, 19934 b; Anton et al., 1996). Urodeles demonstrates a unique ability to regenerate the retina damaged in different ways, including its complete and partial removal, detachment, or cutting of the optic nerve (Grigoryan, 1996). Retina detachment in the newt is an interesting model for analyzing effects of microgravity on neural regeneration in vertebrates. This operation leads to cell proliferation, transformation, and migration both in neural retina @JR)and underlying retinal pigment epithelium (NRP), which provide for neuronal retina restoration (Grigoryan et al., 1996) and, in some cases, for the formation of additional retinal structures (Grigoryanand Mitashov, 1985;Grigoryan, 1992). When developing under conditions of microgravity, these 293
294
E. N. Grigotyan, et al
processes should have certain peculiarities, as it was demonstrated for limb and eye lens regeneration in newts. On the other hand, data on the effect of exposure to microgravity on retina and optic nerve regeneration in vertebrates are virtually absent, although this problem, as well as the general problem concerning the pattern of regeneration processes in vertebrates under spaceflight conditions, has both theoretical and practical significance. Studies on the state of human visual system revealed changes in retinal blood circulation and intraocular pressure occurring under conditions of simulated microgravity (Kuz’min, 1984) and rapidly changing g-loads (Kroll et al., 1987). These microgravitydriven changes may have significance for restoration of the vertebrate eye after neural retina detachment. Hence, studies on the processes occurring under conditions of changing gravity in vertebrate retina restoring after its detachment are quite interesting and important. In this work, we tried to reveal specific morphological features and types of regenerative cell response in the neural retina regenerating after its complete detachment in newts exposed to microgravity simulated by clinorotation.
MATERIALS AND METHODS Newts PZeurode1e.swaiti from the aquarial room of the Institute of Developmental Biology, RAS were used in experiments. The animals were selected by size (100-105 mm) and weight (5.5-6.0 g). Neural retina has been detached in both eyes of the newts nine days before the clinorotation as follows. The lens was first removed from the animals narcotized in physiological saline for amphibia (0,65% NaCl) containing MS-222 (1:3000) through the incision of cornea in the pupil area. Then a slight atraumatic strain was created by slightly pressing of the limb circle by glass rod with a round tip until a moment when folds of detached retina became visible through the pupil. Two animals were injected intraperitoneally with [3HYJ-thymidineat 5 mCi/g (specific activity 4,6 Ci/mM, “Isotope”, Moscow, Russia) nine days after operation (basal or conventional control) and 3 h before fixation. HaIf of the remaining newts were mounted in the clinostat. The other half served as a control (see below). The so-u&d “radial” (radially - oriented) fast rotating clinostat was custom-built in the workshops of the Zoological Institute, University of Cologne, Germany. It consisted of six disks driven by an electric motor (Figure 1). The rotation axis passed through the centers of the disks. Constant lOO?! humidity required by newts was maintained by submerging of disks once per revolution into water that filled l/3 of the clinostat chamber. The animals were fixed on the clinostat disks using elastic cotton net (Hartmann, Germany) in the way that the rotation axis passed aposteriorily through the frontal part of the head where the eyes were located. Insignificant changes in body position were allowed to reduce stress in the animals. Maximal deviation of the newt eyes from the rotation axis was no more than 8 mm, and this caused some gravity vector changes. Our calculations made according to Silver (1976), showed that, at 60 rpm, gravity force varied from 8,05 x lo‘3 to 3,21 x lo’* g. Clinorotation at 60 rpm was performed in the dark at 21-22” C for seven days. Equal number of the operated animals (4 newts) served as a control and were kept under similar Fig. 1. Devices used in the study on conditions in an aquarium, on flter paper saturated with fimctional microgravity influenceupon water. Injections of [31-I+thymidineto animals of both groups neural retina regeneration in the newt were made immediately a&r switching off the clinostat and
Microgravity
295
Effects on Neural Retina Regeneration in the Newt
three hours before fixation. Cell proliferation and morphological changes in different zones of detached retina were studied using autoradiographic, electron microscopic and histological analysis followed by morphometry (for a detailed description of methods see: Mitashov et al, 1987; Grigoryan et al., 1996). Morphometric measurements were made with the aid of ocular micrometer in sagittal sections (5 mkm) stained with nuclear fast red. The state of retina after its complete detachment in the control and experimental newts was evaluated using such criteria as the number of proliferating and degenerating cells, absolute thickness of the retina and relative thickness of its layers, the number of Mtiller cell processes, the number of macrophages, development of the optic nerve, reaction of the retinal pigment epithelium, etc. (Table 1). Measurements were made in the every second or&ird section in a complete series of eye cross-sections representing the central zone of the retina (equivalent to the size of the pupil). Data of measurements and calculations were processed statistically.
Table 1. Criterion chosen for study on simulated microgravity influence upon neural retina regeneration after its detachment in the newt Pleuroakles Walt1 Criterion “Negative” l.Celldeathin NR 2. RPE cell responses (transformation, proliferation and migration) 3. Phagocytic cell reaction “Positive” 1, Development of photoreceptor cell outer segments 2. Thickness of nuclear layers 3. Thickness of plexiform layers 4. Optic nerve development 5. Extension of NR-RPE reattachment 6. Volume of vitread endfeet ofMtiller glial cells 7. Cell proliferation in inner nuclear layer
Experiment
Control
+ + +
++ ++ ++
+t i+ +t
+ + +
U
+
U
+
U
+
U
+
RESULTS Morphological analysis of neural retina atIer its complete detachment in newts demonstmted firstly that the operation procedure was relatively autraumatic. No breaks or cuts in retina and retinal pigment epithelium were observed in eyes of the operated newts during the observation period: neural retina was detached as a whole, and retained partly destructed photoreceptor outer segments. After detachment, the retina survived for a long time: in operated animals that were additionally fixed one month after operation, no signs of retina degeneration were observed. Development of a characteristic responses of eye tissues to the lens removal and retina detachment was observed on the 9th day after the operation (basal or conventional control). Cells of retinal pigment epithelium and iris dediBerentiated and begun to proliferate. A number of [3H]-Tdr-labeled cells in these areas constituted 8 to 12%. An increase in macrophage number was observed near the pigment epithelium, dorsal iris, and in the local areas of cell death, usually deep inside of retinal folds. Some of the
2%
E. N. Grigoryan, et al.
mactophages were labeled. A small number of [jH]-Tdr-labeled cells was also observed in iris and cornea. This also indicates the beginning of post-operation eye regeneration. Lens regeneration was at the I-II st. according to accepted classification of Yamada (1967). The content of labeled cells was quite low in the retina which was thinned and located in the eye cavity forming folds at this time.The labeled cells were localized in the inner nuclear layer (ML) , and very rarely in other retinal nuclear layers (ONL, CCL) and in the area of the optic nerve outlet. Absolute numbers of the labeled cells and their diitribution among different retinal areas after mounting the animals into the clinostat are shown by Figure 2. The following changes in the morphological state and cell proliferative activity of the detached retina have been observed at the 16th post-operation day and after the end of chnorotation and in the ground control. The total number of proliferating retinal cells was lower in both groups of animals than at the 9th postoperation day (basal control). This 2-3 times decrease was similar in all the analyzed areas (Figure 2). Both on the 9th and 16th days post- operation the majority of r3HJ-Tdr labeled cells were associated with inner nuclear layer of the central part of the detached 220 retina. Additionally applied electron microscopy 200 analysis and analysis of semi-thin sections have 180 shown that the population of cells incorporated 180 13Hj-Tdr in inner nuclear layer is represented by I~ Mtillerian glial cells and by interneurons, 120 presumably bipolats. In this case the labeled nuclei 100 were located deep within this layer and had a radial 80 orientation. We could not detect very signifkant 6o differences in proliferative activity of retinal cells in *O ONL INL GCL the control animals and in the animals subjected to n=lOO;N=S clinorotation because of a low general proliferation q CENlR(C) q CENTRQ Doa (C) m oCf=(E) [ fl level in both cases. One can only note that there were more proliferating cells in the central zone of Fig. 2. Absolute number of [3H] -Tdr labeled the retina, and in particular in the inner nuclear cells in dorsal and central parts of detached neural layer of the clinostat -exposed animals (Figure 2). retina a&r chnorotation and in the control Partial cell death occurs in avascular newt retina a&er its complete detachment. It is due to disorders on cell trophy which is carried out under normal conditions at the expense of choroidal circulation. An absolute number of necrotic cells in the detached retina has been counted in 100 central sections on the 9th day _ n=loo;N=l and then on the 16th day post-operation in the SO0 experiment (7th day of chnorotation) and in the control. As follows from the Figure 3, the number 600 of necrotic cells is rather high on the 9th day post400 operation (particularly in the dorsal area of the retina), but it decreases sharply by the 16th day. 200 The most sign&ant decrease in the number of n v-necrotic c4Als occurs in the clinostat-exposed 16VWO* 9.0" animals as compared to the control (Figure 3). l-daytiopemtkw--dayaftwrotation Thickness of the nuclear layers and the whole retina between inner and outer limiting membranes Pig. 3. Absolute number of necrotic cells obaerved 1 different areas of detached neural retina before (excluding the outer segments of the photoreceptor m cells) served as an another criterion of the and a&r clinorotation and in the control morphological state of the detached retina. The total thickness of the detached retina was increased on the 16th day post-operation as compared to the 9th
Microgravity Effects on Neural Retina Regenerationin the Newt
297
day (Table 2). The increase in thickness is due to limited coordinated growth of its nuclear layers where proliferation was detected (Table 2) and to reconstitution of the cytoplasm volume. No preferential growth of a retinal zone was observed. No sign&ant difference in this parameter was observed in the retina of the clinostat-exposed animals as compared to the control ones. At the same time measurement of the total thickness of the morphologically nuclear&e plexiform layers (calculated by subtraction of the total thickness of the nuclear layers from the total thickness of the retina) rev&s their significant increase upon clinorotation at least in the dorsal and central areas of the neural retina (Table 2). Table 2. Total thickness of the detached retina (NR), thickness of its nuclear layers, and relative thickness of plexiform layers (PL) before and after the clinirotation and in the control. Days
9" 0"" 16" 0"" 16" 7'"
92,1*1.9 93.7*1,9 104,2+2,3
28,Ok I ,0 27,l zO,8 28,1*1,1
Dorsal area 35,421,3 3452 1.2 39,3? 1,l
10,4&,5 12,4&,6 11,5&1,4
19.9% 21,1% 24.3%
lO,l?xl,3 13,1&0,5 12,4*,3 GCL
16,6% 25,3% 36.2% PL
centnd area
60,7-c1,4 21.6rO.6 9" 0"' 27,9~1,2 100,6*2,5 16'0"" 27,8ti,9 112,4%2,2 16" 7"" ONL NR -*-days after operation, -**days after rotation.
19,34,7 34,2* 1,o 31.6ti.9 INL
We have also counted a number of long radial processes (accessory prolongations) of the M&&n glial cells in the inner plexiform layer in different areas of the retina. These processes reach towards the vitreous body through the inner part of the retina and can be visualized using NS-9 and ZS-I filters @ST 941 l60). Counting of fibers of these cells in different lP=QN=d n=loop6 F#n;N=8 areas of the retina demonstrates that their number 45 increases by the 16th day post-operation, and that 40 this increase is significantly higher in the clinostat36 exposed animals (Figure 4). Interestingly, a greater 30 increase of the number of processes isobservedin 25 20 the central area of the detached neural retina. We have also analyzed other eye tissues besides the Is above-described studies of changes in the detached lo retina. The changes in morphology of these tissues 5 9v *- daytier cqet~~~ - day alter ;%n may also reflect the effect of clinorotation on ( El DORS q CENTR 61 VENTR ] regeneration processes in the eye. Eye lens regenerates after its removal during the operation. Fig. 4. Number of Miiller glial cell processes in It is formed on the 16th day post-operation from different areas of detached neural retina before and the cells of the distal part of dorsal iris. The after clinorotation and in the control regeneration rate was somewhat higher in the ciinostat-exposed animals than in the control: III-IV vs. II-IV stages respectively. Optic nerve which is retained during the operation was better developed afbx the clinorotation as determined by its diameter in the area of ocular nodule and its morphology in the area of its going out of eyeball. Retinal pigment epithelium undergoes significant changes directed to regeneration of the retina after its detachment In the present work we have chosen time intervals where we could only observe the beginning of this process: cell dedifF&ntiation, cell proliferation and cell transformation into phagocytes as was described earlier (K&e, 1973). These processes were observed both in the control animals and in the
298
E. N. Grigoryan, et al.
clinostat-exposed ones. The differences were found in the extension rate of the pigment epithelium areas exhibiting this response to the retinal detachment. The retinal pigment epithelium areas expressing the morphological changes were much more reduced in the experimental animals as compared to the control. Inversely, the retinal pigment epithelium redifferentiation zones were larger in the clinostat-exposed animals. Cells of redifferentiated retinal pigment epithelium were in close reapposition to photoreceptor outer segments of morphologically normal retina. At the same time we did not measure the size of reattachment regions because of a possibility of artefacts in the morphological pattern of interaction of the retina and pigment epithelium due to relatively harsh fixation conditions. We do not supply data on changes in morphology of photoreceptor outer segments because of the same reason. We have counted the number of macrophages in the eye cavity near the detached retina and within its layers before and after clinorotation, and in the controls (9th and 16th day post-operation, respectively). A significant number of macrophages were observed before the clinorotation (50-300, SO-300 and 50-150 in the dorsal, central, and ventral areas of one eye, respectively). The number of macrophages decreased right afler the clinorotation and constitutes 5-50, 5-60, and 5-40 in the same zones, respectively. At the same time a number of macrophages was somewhat higher in the eye dorsal and central zones of the control animals: 5-150, and 5-200, while in the ventral zone it was similar to that of the experimental animals (530). DISCUSSION Our data suggest that exposure to microgravity simulated by clinorotation at 60 rpm may cause a number of changes in the morphological state of retina after its detachment. According to the chosen criteria (Table 1), these changes concern largely the development of the plexiform layers of the detached retina, Mtiller glial cells, and, to a lesser extent, the number of macrophages, proliferating and degenerating retinal cells, and the ability of the retina to restore apposition to RPE. They occurred mainly in the dorsal and central zones of the detached retina. We found that an increase in thickness of the retina, resulting from growth of its reticular layers, in newts subjected to clinorotation for seven days is greater than in control newts kept in an aquarium. The plexiform layers appear to grow due to an increase in the number and length of neuronal and glial cell processes, which form these layers. Mtiller glial cells, located radially in the inner nuclear layer and representing the major glial component of the retina, belong to this growing population of cells. Published data indicate that these cells play multiple roles in the retina. They can easily change their phenotype and undergo biochemical changes depending on the state of retinal neurons and their relationships (Moscona, 1987). Mtiller cells can synthesize various macromolecules supporting neuron functioning and maintaining extracellular microenvironment during prolonged retinal detachment (Lewis et al., 1989). These nonneuronal cells are a structural component of the adult retina and play a morphogenetic role in the developing retina (Wolburg et al., 1991). Finally, they supposedly play the role of macrophages, being the major retina “cleaners” (Nishizono et al., 1993), and are responsible for myelin production in the fibrillar layer of the, optic nerve (Huges and La Velle, 1974). All this suggests that Mtiller cells maintain the retinal homeostasis. Published data and our results show that all the above functions become activated after retinal detachment. Morphologically, it is manifested in cell proliferation, hypertrophy, migration, and nuclear translocations (Guerin et al., 1990; Grigoryan et al., 1996). These processes are a typical cell response to changing environment and may be activated by factors other than retinal detachment. In our experiments, the loss of topological and functional relationships between the retina and RPE was accompanied by changes in gravity caused by clinorotation. Hence, the regenerative response of Miiller glial cells to retina detachment, manifested in an increase in the number of accessory processes, was enhanced in this case by altered gravity. These data agree with the results of our study on cell proliferation in detached retina: although the level of proliferation in the latter was generally low, newts subjected to clinorotation demonstrated an increase in proliferative activity of cells of the inner nuclear layer in the central zone of
Microgravity
Effects on Neural Retina Regeneration in the Newt
299
the retina (Figure 2). Data on radial orientation of [3H]-TdR-labeled nuclei and morphology of labeled cells in ultra- and semithin sections of the retina, which resembled that described by Miiller (1862) and Uga and Smelser (1973), suggest that Mtiller glial cells make a significant contribution to the pool of proliferating cells in the retinal inner layer. These cells were activated for proliferation first by retinal detachment and then by clinorotation. Hence, clinorotation-simulated microgravity may activate numerous natural functions and change the behavior of retinal glial cells after retinal detachment, and this cell response leads to more successful structural and functional regeneration of the neural retina. We found that simulated microgravity affects other parameters of retina regeneration. According to our data, the number of retinal cells degenerating after detachment in newts subjected to clinorotation is much less than in control animals. The same concerns the number of macrophages in the retina and around it. Macrophages appear in the eye cavity in response to eye damage and reflect a long-term (in poikilotherms) inflammatory reaction (Mitashov et al., 1979). Disappearance of macrophages indicates cessation of this reaction and the absence of additional signals for macrophage migration into the eye cavity. According to this criterion, simulated microgravity appears to have a favorable effect on regeneration of the detached retina. Better development of the optic nerve, a higher rate of lens regeneration, and behavior of RPE cells in experimental animals provide additional evidence for this conclusion. Most of the above-mentioned differences in the state of detached retina in animals subjected to clinorotation, activation of Muller cell growth in particular, were better manifested in the dorsal and central areas of detached retina rather than in its ventral part. The adaptive differences in growth capacity of Muller cells between the central and peripheral zones of the retina were described previously and attributed to the fact that thickness of the retina decreases toward the eye growth zone (P&a et al., 1989). However, our previous experiments on eye lens regeneration under microgravity simulated by clinorotation (Anton and Grigoryan, 1993a) also demonstrated preferential activation of proliferation in tissues of the dorsal eye area (eyelid skin, iris, and cornea) These spatial differences are attributed either to intrinsic peculiarities of cells located in different zones of a certain tissue or to peculiarities of their interactions with surrounding cells and tissues. The latter explanation seems likely. Although the stimulatory effect of microgravity on lens and limb regeneration in newts was first demonstrated in 1985 (Mitashov et al., 1987) and has been studied since then from different aspects under the actual spaceflight conditions (Grigoryan et al., 1992, Mitashov et al., 1990, 1996) and in experiments with simulated microgravity (Anton and Grigoryan, 1993a,b; Anton et al., 1996), mechanisms responsible for the effect of microgravity on regeneration processes remain unclear. In the case of retina regeneration, microgravity may affect the expression of known growth-stimulating factors, their circulation in eye cavity or transport to target ceils. For example, recent experiments revealed tissuespecific changes in the level of TGF-b mRNA in skeletal tissues of rats growing under spaceflight conditions (Westeriind and Turner, 1995). Microgravity can also specifically alter cell adhesion in the retina, whose role in triggering retina regeneration is currently being investigated (Raymond et al., 1995). Moreover, studies on urodeles exposed to spaceflight conditions revealed profound changes in their calcium metabolism (see: Berezovska et al., this volume), and calcium ions are known to play an important role in the transduction of hormonal and neuronal signals. This suggests the involvement of microgravity-induced calcium changes in stimulating regeneration of neural retina in the newt. A general increase in the level of retinal cell metabolism resulting from an adaptive reaction (generalized stress) of the whole organism to altered gravity may also stimulate this process. These ideas require further experimental confirmation. In subsequent experiments, we plan to use other models of neural retina regeneration in newts (in particular, regeneration after cutting the optic nerve and blood vessels) for analyzing the role of effects caused by microgravity in neural regeneration in space.
300
E. N. Grigoryan, er ai.
ACKNOWLEDGMENTS We are grateful to V. A. Poplinskaya for her technical assistance in processing experimental material. This work was supported in part by DFG and by contract NAS 15-10110. REFERENCES Anton, H. J. and E. N. Grigoryan, Simulated Microgravity Induces Increase of Regeneration Rate, Cell Proliferative Activity, and Enhancement of Size of Regenerate during Wolffran Lens Regeneration in the Newt. 86. ye&. Deutsche Zooi. Ges. 1993. Salzburg (Kurzpublikationen), 86,204 (1993a). Anton, H. J. and E. N. Grigoryan, Altered Influence of Gravity Can Provide Long-term Effect on Forelimb and Tail Regeneration in the Newt. (Preliminary Results of Experiments on Simulated Microgravity), 86 Vehr. Deutsche Zool. Ges. 1993. Salzburg (Kurzpublikationen), 86,203 (1993b). Anton, H. J., E. N. Grigoryan and V. I. Mitashov, Influence of Longitudinal Whole Animal Clinorotation on Lens, Tail, and Limb Regeneration in Urodeles, A& Space Res., 17, (6/7) 55 (1996). Berezovska, 0. P., N. V. Rodionova, E. N. Grigoryan et al., Effect of Microgravity on Activity of Osteoclasts in Newts, Adv. Space Res., (in press). Grigoryan, E. N. Eye Regeneration after Retina Detachment and Tear-off in the Newt Pleurodeles waltl, Bulletin of Russian Acad Sci., ser. Biol., N 1, 18 (1992). Grigoryan E. N., Retina of Urodeles as a Model for Study on Retinal Regeneration Capacities in Other Vertebrates, Ontogenez, 27, N 3, 173 (1996). Grigoryan E. N. and V. I. Mitashov, Cultivation of Pigment Epithelium in a Cavity of Lens-ectomized Newt Eye, Ontogenez, 16, N 1,34 (1985). Grigoryan, E. N., I. P. Ivanova and V. A. Poplinskaya, Identification of a Novel, Intrinsic Cell Source for Neural Retina Regeneration after its Detachment in Newts. I. Morphological and Quantitative Studies. Bulletin of Russian Acao! Sci., ser. Biol, , N 3, 3 19 (1996). Grigoryan, E. N., E. A. Oigenblick, S. Y. Tuchlcova et al., The Influence of Space Flight Factors on Lens and Limb Regeneration in Newts, in Results of Investigations aboard Russian Biosatellites, pp. 345350, Nauka, Moscow (in Russian) (1992). Guerin, Ch. J., D. H. Anderson and S. K. Fisher, Changes in Intermediate Filament Immunolabeling Occur in Response to Retinal Detachment and Reattachment in Primates, Invest. OphthaZ. Vis. Sci., 31, N 8, 1474 (1990). Hughes, W. F. and A. La Velle, On the Synaptogenic Sequence in the Chick Retina, Anat. Rec., 179,297 (1974). Keefe, J. R., An Analysis of Urodelian Retinal Regeneration: III. Degradation of Extruded Melanin Granules in Notophthalmus viridescens, J. Exp. Zoof., 184,233 (1973). Kroll, G., J. Draeger, H. Brande, et al., Untersuchungen uber den Intraocularen Druck bei rasch wechselnden g-Belastungen in der Z-Achse, Mitt. Dtsch. Ges. Luft-und Raumfahrtmed, 2, 10 (1987).
Microgravity Effects on Neural Retina Regeneration in the Newt
301
Kuz’min, M. P., Blood Circulation in the Human Eye Retina and Eye Pressure in Conditions of Simulated Zero Gravity, in Space Anthropology Techniques andMetho& of Stuc&, p.447. Nauka, Leningrad, (in Russian) (1984). Lewis, G. P., P. A. Erickson, C. J. Guerin, et al., Changes in the Expression of Specific Mueller Cell Proteins during Long Term Retinal Detachment, Exp. Eye. Res., 48,93 (1989). Mitashov, V. I., V. I. Starostin, A. I. Sludskaya, et al., An Autoradiography Study of Macrophagal Activity during Eye Regeneration in Adult Newts, Ontogenez, 10, N 4,365 (1979). Mitashov, V. I., E. N. Grigoryan, S. Y. Tuchkova et al., Organs and Tissue Regeneration in Amphibia under the Space Flight Conditions, in Life Science Research in Space. pp. 299-303, ESA and ESTEC Publ. Division, Netherlands (1987). Mitashov, V. I., E .N. Grigoryan, S. Y. Tuchkova, et al., Lens and Limb Regeneration in the Newt during and a&r 13 Day Long Space Flight, in Microgravity is a Tool of Developmental Biology, ESA and ESTEC Publ. Division, Netherlands, 85 (1990) . Mitashov, V. I., N. V. Brushlinskaya, E. N. Grigoryan et al., Regeneration of Organs and Tissues in Lower Vertebrates during and after Space Flight, A&. Space Rex, 17, (6/7) 241 (1996). Moscona, A. A, Role of Cell Contact in Phenotype Stability and Modification: Studies on Retina Cells, Amer. Zool., 27, 137 (1987). Mtiller, H., ijber das Auge des Chamiileons mit vergleichenden Bemerkungen, Zietschz?., 3, 10 (1862).
Wiirzb. Naturw.
Nishizono, H., Y. Murata, M. Tanaka et al., Evidence that Mueller Cells Can Phagocytize Egg-lecitincoated Silicone Particles, Tissue and Cell, 25, 305 (1993). Prada, F. A., M. M. Magalhaes, A. Coimbra et al., Morphological Differentiation of the Mueller Cell: Golgi and Electron Microscopy Study in the Chick Retina, J. Morphoi., 201, 11 (1989). Raymond P.A, J.E. Bra&d, J. K. Knight et al., Role of Cell-cell Interactions in the Regulation of Retinal Regeneration, in Publ. of 6th Internat. Sjmp. on Neural regeneration, Asilomar, USA, 29 (1995). Silver, I. L. The Dynamics of a Discrete Geotropic Sensor Subject to Rotation-induced Gravity Compensation, J. Z’heor.Biol., 61,353 (1976). Uga, S., and G. K. Smelser, Comparative Study of the Fine Structure of Retinal Mueller Cells in Various Vertebrates, Invest. Ophthalmol., 12,434 (1973). Westerlind, K. C. and R. T. Turner, The Skeletal Effects of Spaceflight in Growing Rats: Tissue-specific Alterations in mRNA Levels for TGF-beta, J. Bone andMineral Rex, 10,843 (1995). Wolburg, H., E. Willbold, and P. G. Layer, Muller Glia Endfeet, a Basal Lamina and the Polarity of Retinal Layers Form Properly in vitro only in the Presence of Marginal Pigment Epithelium, CeN Tisnre Res., 264,437 (1991). Yamada, T., Cellular and Subcellular Events in Wolffian Lens Regeneration, Curr. Top. Develop. Biol., 2,247 (1967).