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Influence of longitudinal whole animal clinorotation on lens, tail, and limb regeneration in urodeles
Adv. S/me Res. Vol. 17, No. 6/7. pp. (M7)55-(M)6S, 1996 Copyright Q 1995 COSPAR
Printedin Great Britain. All rights reserved. 0273-l 177196$9.50 + 0.00
INFLUENCE OF LONGITUDINAL WHOLE ANIMAL CLINOROTATION ON LENS, TAIL, AND LIMB REGENERATION IN URODELES H. J. Anton,* E. N. Grigoryan**
and V, I. Mitashov**
** Instituteof ~e~e~o~~e~~~Biology,RussianAcademy Science, Vavib Street 26, 117808 Moscow,Russia
ABSTRACT Two species of newts (Ur~~e~a) and two types of clinostats for fast clino~~tion (40 rpm) were used to investigate the influence of simnlated weightlessness on regeneration and to compare results obtained with data from spaceflight experiments. Seven or fourteen days of weightlessness in Russian biosatellites caused acceleration of lens and limb regeneration by an increase in cell proliferation, diffemntiation, and rate of morphogenesis in comparison with ground controls. After a comparable time of clinorotation the results obtained with T~~~~ ~lgar~~ using a horizontal clinostat were similar to those found in spaceflight. In contrast, in Pleurodeles wdtl using both horizontal and radial clinostats the results were contradictionary compared to Triturus. We speculate that different levels of gravity or/and species specific thresholds for gravitational sensitivity could be responsible for these contradictionary results.
The optimal answer to an injury in living systems is perfect regeneration of the missing parts. in most vertebrates posttraumatic regeneration capacity is negligible. However, in urodeles ~~rn~~i~j~}, regenerative potencies are not restricted to physiologic mg~em~on and wonnd healing only, but also involve the capability to develop perfect organ and body parts similar to the ontogenetieally formed originals. Eye, limb, and tail are those body parts which best form externally visible regenerates. These systems are very well described 111,/2/. Regeneration experiments in space were first proposed in 1984 /3/,/4/, and first carried out in 1985 aboard a Russian BIOCOSMOS satellite followed by six further spaceflights focusing on regeneration during the last decade. It was shown that when lenses were removed and limbs were amputated, ~gene~on was generally compile in both sp~~i~t weightlessness and lg ground controls. However, in all expe~men~, space-related factors provided specific stimulative effects, such as acceleration in regeneration rate and differentiation /5/, /6t, 171,/Si, 19/. Moreover, for eye regeneration, a remarkable enhancement of size of lens regenerates was found after spaceflights of animals whose lenses had been removed one week before start 151, 161, 171, 181. Addition~ly, abno~~ shares of the newly formed lenses (e.g., cataracts) and of regenerated forelimbs (e.g., skeleton malformation) were also observed, possibly related to other spaceflight factors than weightlessness /5/, 161,i7l. By advancing the animals to various regeneration stages before start, it was possible to determine the most sensitive stages of lens and limb regenemtion. It was shown that spaceflight effects occur mainly at early stages of lens and limb regeneration /6/. It was also observed that the influence of spaceflight on cell proliferation and differentiation has long-term character /9/. The spaceflight factors in~uen~ing regeneration are not only weightlessness, but also changes in light/dark cycle, temperature, low dose irradiation, vibration, and short term hypergravity during
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1~~~ and landing time. The infIuenee of such factors on mgene~on in urodeles as temperature flO/, fll/, /12/, season 1131, body size and age 1141, x-rays /15/, 1161,f171, 1181, /19/, and ligh~d~ cycle /20/,/21/,/22/, was described earlier under ground laboratory conditions. In order to better understand the specific influence of weightlessness on regeneration, all known or expected changes in Ytiors taking place in space were simulated as best as possible parallel to each spaceflight in ground control experiments /5/, lb/, /‘J/, ISI, 191. Among the different means to study the effect of gravity alterations, the effect of fast clinoro~on was used to investigate the influence of novel gravitational fields on different biological models such as cell cultures, developing animal embryos, and plants /23/, /W, 1251. To investigate the influence of low gnwity on adult urodeles, the main objectives of the present work were: 1) to develop adequate conditions to simulate microgravity in experiments with urodeles, 2) to find the effects of sirn~~ micro-g on regeneration in urodeles and 3) to compare the results with those which have been found in space experiments aboard the BIOCOSMOS satellites. MATERIALS AND METHODS Instruments and Conditions Two types of fast rotating clinostats were used. The first type was equipped with two ‘horizontally-oriented’ cuvettes ~O~~no~t~ (fig. 1, left) in which the rotation axis run through the cuvette center. Newt’s cranio-caudal axis was aligned as well as possible in the center, parallel to the rotation axis. Speed of cuvettes was 60 rotations per minute according to calculations showing that at this speed low g levels, i.e., mostly microgravity is reached. Humidity-satumtion of surrounding was achieved by placing a dish, approx. 300 cntz, filled with distilled water, on the bottom of the clinostat box. During rotation the ~~~d~ cycle was 0124 h; however7 5 min of lighting per day were allowed to check the animal’s state and the exposure conditions. The temperature was 2&l°C. The second type of clinostat (fig. 1, right) had six radially-oriented plates with horizontally oriented rotation axis (RO- clinostat). The correct position of the newts on the plates was reached by putting them into an elastic knit cotton mantle (Stiilpa/Hsrtmamr) fixed by clamps at the edge of the plates. Iu this way the cranio-caudal axis of the animals coincided with plate radius, but regenerating forelimbs and eyes were located in the center of rotation. The bottom of the clinostat box was filled with tap water approx. 2 cm in depth. The rotating plates had steady contact with water at their periphery, and the ends of the cotton mantles dipped in it during each rotation. This guaranteed 100% humidity inside the clinostat box, and moist surface of animals, thereby protecting the skin from drying. Plate speed, temperature, and the light/dark cycle were the same as in the HO- &no&t. Fig. l.(lef?) horizontally oriented (HO-) clinostat (Briegleb) and (right) radially oriented (RO-) clinostat (Anton) In the experimental controls, the same conditions were maintained, except clinorotation (fig. 1) and resembled those aboard the biosatellites, In the HO- clinostat the maximum possible distance of any body parts from the rotation center ranged from 5 to 8 mm for Pleurodeles and from 1 to 2 mm for ?“turus. In the RO- clinostat, the head and forelimbs were not farther from center than in the HO- clinostat, but for the other body parts, e. g., hind limbs and tails there was exposure to increasing gravity vectors. The HOclinostat was preferred, because it provided much less mechanical noise than the OR-clinostat. Animals Adult ~~~~rodel~~ ~a~~~(total length 100 to 110 mm and weight 5 to 6 g respectively, bred at the fnst. of Dev. Viol. Moscow) and T~~~~ vulgaris (t&al length SO-55 mm and weight lg approx., bred at
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Zool. Inst., Univ. of Cologne) were used in our experiments. The latter was used for the most part because of its low size and weight, Experimental procedures ln accordance with the procedures of the spaceflight experiments f5f, 161,/7f, 181,the animals underwent operation 5-7 days before clinorotation. After anesthesia (MS 222 1:2000), lenses were microsurgically extirpated from both eyes through a small comeal incision, both forelimbs were amputated at the proximal ~ugopo~~ (forearm) level, and tail halfway between cloaca and tail tip, i.e., regeneration always took place simultaneously in three different body parts under ‘multiple lesion conditions’. Experimental and control animals underwent operation at the same time and were kept before clinorotation under similar conditions (21& lo C, light/dark cycle 12/12 h). The experimental animals were put in place in both HO- and OR-clinostats just before the beginning of the rotation period of 7 or 14 days. During this time the animals were not fed (to correspond with the space flight conditions).Upon removal from the clinostats, all animals were healthy. As a behavioral response to change of ‘gravity field’, they moved backwards for some minutes. To investigate the cell proliferation rate, some experimental and control animals were injected ~~e~~ne~ly with 3H-thymidine immediately after clinorotation and several others one week later (5lKi/g body weight, ‘Isotope’, Moscow). After four hours of incubation, the animals were anesthetized, sacrificed and samples (eyes, limbs, and tail) were fixed in Bouin’s fixative solution. From the remaining animals, the fully regenerated lenses were reextirpated and forelimbs and tails reamputated. The following second regeneration period was also observed. The regenerative progress was ascertained by determining the stages of lens /26/ and forelimb regenerates and by measuring the size of lens and tail regenerates /27/,/28/. For skeleton analysis, forelimb regenerates were stained ‘in toto’ with Vista-blue. Histologic~ and autor~og~hic~ analyses were performed in accordance with accepted methodsi51. RESULTS Lens regeneration in newts Tritmwsexposed in HO- clinostat for 14 days No notable differences in proliferative activity and stages of the lens regenerates were found at starting time: cell proliferation in the imrer layer of dorsal iris (source of lens regeneration) was initiated CH labeling index = 3 to 5%) and the regenerates achieved stages I-II both in experimental and in control animals. Just after the end of the rotation period, regenerates in experimental and control groups were advanced and showed non-synchronous stages IV-VIII. However, in comparable stages, the proliferation rate of iris cells was 1.3 - 1.5 times higher than in the controls (fig. 2a). This increase was also observed in tissues not involved in lens fo~~on (e.g., epide~~ cells of the dorsal eye lid). Ad~tion~ly, in one of 20 eyes of the clinorotated animals, a voluminous area of pigmented epithelial cells had developed, instead of a lens regenerate. Histologically, this area was characterized by numerous mitotic figures and the majority of cell nuclei had increased in volume (S-, G2-phase). Obviously, a melanoma had developed (fig. 3). A rn~i~~t growth of this type occurs very seldom in newts and was not found in any of our control and space flight experiments with Pleurodeies. The loss of proliferation control was possibly associated with the induced increase of cell proliferation.
orig con exp
con exp
b Fig. 2. ;a) 3H-Td labeling index of lens regenerates at start (stage 11)and 14 days’later at end (stage VII) of clinorotation; (b) mean diameter of original, control and clinorotated lens regenerates 1. reg. = one month, 2. reg. = two and half months after ending rotation, AU = arbitrary units; (c) projection of lens regenerates (mean volumes);
b
a
C
e d Fig, 3. Lens regeneration in Trifurus. Top: whole eyes; (a) control; (b) experimental animal. Note the opaque anterior eye chamber; (c) e~e~rnen~ animal with melanoma fo~ation. Note the prodding cornea. Bottom: histology; (d) median section of the right eye. Note the connection of the tumor with the dorsal iris margin; (e) higher m~nifi~ion. C = cornea, NR = nenral retina, RP = retinal pigment epithelium, T = tumor, I = upper and lover iris margin, p = pigment gran~es, t = mitotic figures
Fig. 4. Extirpated lenses (Trifzms) (a) original lens, @) newly formed control lens, (c) newly formed experimental lens. a
b
Fig. 5. Forelimb regenerates (a, b) just after clinorotation (c, d) three weeks later. ‘lbe experimental regenerates (b, d) are more advanced than the corresponding control regenerates (a, c). a
d
b
Fig. 6. An~po~~ skeleton rn~fo~~ons in limb regenerates of clinorotated animals (a) normal, Co), (c), (d) different grades of skeleton malformations. a
b
d
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Two months after clinorotation, the fully differentiated first lens regenerates were reextirpated and a second regeneration was initiated. Tbe mean lens diameter of the first experimental regenerates was approx. 110% the volme of that of the controls. The second lens regenerates, extirpated and measured 6 weeks after the first reextirpation, showed comparable differences in size (fig. 2b, c; 4). Forelimb regeneration in Triturus exposed in HO-clinostat for 14 days Forelimbs were amputated seven days before start of clinorotation. At start, wound healing had finished and the epidermal cap was formed in experimental and control groups. At end of clinorotation, the forelimb regenerates of rotated animals were markedly advanced, having reached the stages ‘cone’ to ‘palette’, whereas at the same time, control regenerates achieved stages ‘bud’to ‘cone’ only. Some typical differences between experimental and control regenerates are shown in figure 5, The proliferation rate t3HTd-labeling index) of the blastema cells of the experimental regenerates of corresponding stages ‘late bud’ and ‘early cone’ had advanced up to 1.4 and 1.3 fold, respectively, than those of the controls. Growth and morphological differentiation of the rotated regenerates were accelerated up to the ‘late 3 digit’ stage (fig. 7a).
a
b
regeneration stages
regeneration stages
days after landing
weeks after reamputation
Fig. 7. (a) first and fb) second forelimb ~genemtion of ~r~~~r~svdgaris at& 14 days of clinorotation; C = control, E = clinorotated limbs, d = days, w = weeks, % = percentages of stages at different time.
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Two months after ending rotation, the regenerates were reamputated and the following second regeneration was screened again; it showed that acceleration in the experimental group still continued for up to three and half months (fig. 7b). In contrast with the results obtained in the spa&light experiments, in which an increased number of skeleton malformations among the regenemted forelimbs was observed, the limb regenerates of the clinorotated animals showed no notable differences in frequency and range of abnormalities (see table and fig. 6a, b, c, d).
Table: Skeleton malformation in Triturus forelimb regenerates. group are statistically not significant.
Differences
in experimental
and control
Tail regeneration in Triiurus exposed in HO-clinostat for 14 days Similar to forelimb regeneration, the tail regeneration rate in the clinorotated newts was also higher at all time points. Just before start, the tail stumps of all animals were in the same stage: the wound was closed, an epidermal cap had formed and under it dedifferentiation of dermis, musculature, vertebra, and spinal cord had set in. After ending rotation, the tail regenerates were essentially advanced and reached stages IV - V in the experimental group, in contrast in the control group stages III-IV. In order to allow the tails to grow further, cell proliferation rate was not analyzed at these stages. But some weeks later the absolute length of the regenerated tails and the ratio of regenerate/whole tail length demonstrated the higher growth rate in the experimental series (fig. Sa, b). 100
a Fig. 8. Tail regeneration
con
exp
b
con
exp
in Trim-us (a) tail length (arbitrary units), (b) length ratio (reg./whole tail)
Regeneration in Pleurodeles exposed in HO- and RO- clinostat As a consequence of the small size of Tritums, its position in the HO-clinostat was very close to the rotation axis: the longest distance of animals periphery from center was 1 to 2 mm, so that calculated g ranged between 4.0x10-3 to 8.0~10~~ g (fig. 9a). On this basis, we assume that this experimental series provided a good parallel with real space flight conditions in the Biocosmos satellites. Since spaceflight experiments have been carried out with Pleurodeles, we were also interested in studying the influence of clinorotation on regeneration in this species, especially due to the larger size of the animal, the simulated low gravity was higher than for Triturus. Additionally, in the RO-clinostat the animals were ‘radially-oriented’, so that only the area where regeneration takes place was at the rotation center.
0,30 ________:___-____;;,~--_I_ .~~~~ 'g 0,20 -- -_____:;;~__~___.i___ 2 012
3
4
5 6
7
8
910 mm
distance from center
0,lO - - _,,_d'_____________-___ 012
3
4
5
6
7
distance from cemz
8
910
cm
b a Fig. 9. Changes of calculated gravitational fields relative to the rotated animals: (a) HO- clinostat; (b) RO- clinostat. Lens, forelimb, and tail regeneration in Pleurodeles exposed in HO- clinostat for 14 days The distance of the regenerating parts of PZeurodeles from rotation axis rauged between 5 and 10 mm; i.e., the calculated g-forces varied from 3.2x10-2 to 4.O~lO-~g( fig. 9a). It was surprising that under these low gravity conditions lens, forelimb, and tail regeneration rate was reduced. Before start of clinorotation, histological analysis of all lens regenerates showed stages I to II. After rotation, lens regenerates had achieved stages III to V and the ~s~b~on was relatively similar in both groups. However, 10 days later differences between experimental and control groups were notable: progress in regeneration of the clinorotated animals was reduced and showed, stages III to VII, more divergence than the control regenerates, which during the same period had advanced to stages VII to IX. In forelimb and tail regeneration, analyzed by the same methods as for Triturus, the rotated Pleurodeles showed a comparable reduction as seen in lens regeneration. Lens and forelimb in Pleurodeles exposed in RO- clinostat for 7 days Since in the RO-clinostat only the upper part of the newt’s body was exposed in the area of low gravity simulation, only lens and forelimb regeneration was investigated (tails were not amputated). In spite of this, the peculiarities found in lens and limb regeneration reflected those observed in the experiment with PZe~rodeles rotated in HO- clinostat; they suggest retarded lens and forelimb ~gene~on as well. It should be emphasized that only head and forelimbs were influenced by low gravity forces {approx. 10m2g).The other parts were exposed to conthmously increasing gravity (up to approx. 0.4g ) {fig. 9b). DISCUSSION AND FUTURE ACTIVlTIES The results of our investigations show a clear effect of simulated weightlessness on regeneration in urodeles, in particular, on cell proliferation, tissue growth, and organ regeneration rate. In all series of experiments we found clear, lasting responses which were similar in all studied organs (eye, limb, tail), but varied in dependence on the level or quality of simulated gravity reduction and animal species. The retarded cell p~life~on and regeneration rate in Ple~rode~e~ under conditions of clinoro~on was unexpected, given the results obtained in all space fight experiments (/5/,/6/~7/,/8~. However, the lowest level of gravity that we achieved under the conditions employed was approx. 10-z g. Thus, the ‘slowing down’ of regeneration of the rotated Pleurodeles may have been induced by this. There is no information in literature on the effect on multicellular organisms of long-term exposure to gravitational fields between less than lg and far above ‘micro-g (10-6g). Our data may be the first of this kind. However, we should not discount other factors which could possibly induce the described retardation, e. g., mechanical ‘noises’ produced by rotation of the relatively big and heavy animals (especially in the case of RO-clinostat). In contrast to Pleurodeles, the calculated gravity in the experiments with Triturus was lower. It reached IO3 to 10m4g(HO-clinostat). Precisely these g values were suspected to be a possible threshold of sensitivity of biological processes to acceleration 1241. So, it is reasonable to assume that Trj~~~ rotated 14 days in HO-clinostat lived under ‘sub~~shold acceleration’ or ‘funotional weightlessness’ conditions /29/, 1301. As Briegleb 1241 pointed out, most sensitive acceleration processes may have a threshold far above 10m4gand evidently there are thresholds near lg or above it. However, in the absence of any quantitative data on the effect of acceleration on different
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animals, whether from our own experiments or from literature, we can specuiate that the predominantly water-living Pleurodeles and the predominantly land-living Trifurus could express difibrent ‘sensitivities’ to acceleration. Our results support the hypothesis that moderate alone weigh~essness stimulates regeneration, whereas low-gravity fields higher than 1Q3g could have an inhibitory effect on regeneration. It is also possible that g vectors higher than 10e3gdo not have any effect at all and that ‘big’ newts, Pleurodeles, are more sensitive to mechanical noises (e.g., vibration) produced by the rotation, From experiments with bacteria in Dl and D2 spaceflight missions, it is known that different culture media can be responsible for contradictory results, a phenomenon that has been confirmed by clinostat e~e~rnen~ 13II. The effect of fimctional weightlessness was independent of other environmental conditions. More rapid cell proliferation and tissue growth during regeneration in Trifurus under the conditions observed in spaceflights (BIOCOSMOS satellites), were actually caused by weightlessness, and not by other spacerelated factors. Acceleration of cell m~tipli~on under simulated and real ‘micro-g’ represents our main finding, it is a crucial event in the regenerative processes, and the prerequisite for subsequent differentiation and morphogenesis, which are strongly coordinated and programmed. From current knowledge, cell proliferation in animals is controlled by mechanisms and factors, e. g., well determined growth factors (cytokines), cell microenvironment, cell shape changes, cell to cell and cell to substrate contacts, individual cell gene expression. Our experiments have demonstrated that the mechanisms which regulate cell proliferation under normal gravity, also work under novel gravitational conditions: They also suggest that the basic mechanisms of cell control such as receptor binding and signal transduction are not, in principal, altered by gravity. In experiments in which the effect of simulated ‘micro’- and ‘hypergravity’ on different cell types ‘in vitro’ were studied, it was shown that moderate novel gravity has an effect at particular steps in the signal transduction cascade, resulting in altered patterns of gene expression and ultimately in DNA replication and cell division 129, /32/, /33/. As a result it was suggested that pm~fe~on of cells cultured in a h~ergm~~on~ field could be increased /341, but under microgravity conditions it is markedly decreased 1351. This suggestion is based on data obtained for normal and malignant cells, mostly mammalian, ‘in vitro’, and points to a direct influence of gravity changes on cell proliferation regulatory mechanisms. This is why our data obtained in (multicellular) adult vertebrates (newts) exposed to simulated weightlessness and data obtained on cell cultures cannot really be compared. Moreover, we believe that in our case the influence of wei~~es~ess, either simulated or in space, is first mediated by low-g reactivity of the whole organism. This reactivity, that could be named ‘microgmvitational stress’ is still far from being completely understood, Our work presents evidence on the regulatory role of common integrating systems in organisms, one of them being long-term augmentation of lens, forelimb and tail regeneration rate, after relatively short (7 to 14 days) exposure to real and simulated weightlessness: 1.) three months after return to normal 1 g conditions, the higher proliferation and differentiation rate continued, demonstrated by the first and second ~gene~on; 2.) in Trials an increased mgene~ion rate was found in all regenerating systems analyzed (eye, limb, tail); 3.) the increased cell proliferative activity determined in epidermis (eye lids) which was not involved in eye regeneration. From our results, the hypothesis that gravitational stress is nonspecific becomes improbable. But we do not want to definitively reject this idea, since it seems reasonable to assume that rapid rotation of animals can produce stress factors. We have shown that the reaction of cells to wei~~essness is longterm. However, reversibility has not been shown to date. It could be that when normal gravity is ‘switched off, cells forget or lose the mechanism which was a prerequisite for survival at the beginning of evolution. This mechanism never needed to be protected until the beginning of the spaceflight era. Because of this, there was no necessity to develop a kind of ‘switch on’. The energy freed by the switch off process could be directed to accelerate the regeneration process. There is one indication from our results that cell prolife~on under normal lg conditions is less likely to degenerate than under conditions of weightlessness: the single case of melanoma formation (a very rare event in newts), which we found in the experimental group of 10 animals with 20 lens regenerates. As an important result of our experiments, we have shown that small, lower vertebrates, especially land-living animals such as Tribes are suitable for experiments under real and clino~t-sim~ated
ciinostal Iofluence 081 Organ Regeoeration
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spaeeflight conditions, much better than cell or tissue cultnres. The advantages are: 1.) the living system itself, which guarantees stable conditions during the whole experiment; 2.) artificially induced processes which cause natural reactions with no need for artificial support; 3.) within certain limits the animals need no food aud special care to survive and regenerate (one month and more); 4.) independence of animal survival from fluctuation of temperature in limits of 0 to +28’C (poikilothemral animals); 5.) multiple surgery and regeneration (lens, tail, limb, gu& brain, spinal cord, peripheral nerves) without excessive stress. 6.) the animals are suitable as a system of cell and tissue cultures, i.e., carrying transplants in body cavity, eye chamber etc. Given these advantages, many questions concerning the influence of novel gravitational fields can be tested in clinostat experiments.
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33. M. Limouse, S. Manie, I. Konstantinova, B, Ferrua, L. Schafk, cell activation in microgravity. l&p. Cell Re.s&J 82-86 (1991).
Inhibition of phorbol-ester induced
34. A. Tschopp, A. Cogoli, Hypergravity promotes cell proliferation. Experientia, %1323-1329 (1983). 35. F.K. Chntider, A. Cogoli, Cultiv~on of single ceil in space. ~ppZ.microgr~~ (1988).
tech., 1,115-122.
Printedin Great Britain. All rights reserved. 0273-l 177196$9.50 + 0.00
INFLUENCE OF LONGITUDINAL WHOLE ANIMAL CLINOROTATION ON LENS, TAIL, AND LIMB REGENERATION IN URODELES H. J. Anton,* E. N. Grigoryan**
and V, I. Mitashov**
** Instituteof ~e~e~o~~e~~~Biology,RussianAcademy Science, Vavib Street 26, 117808 Moscow,Russia
ABSTRACT Two species of newts (Ur~~e~a) and two types of clinostats for fast clino~~tion (40 rpm) were used to investigate the influence of simnlated weightlessness on regeneration and to compare results obtained with data from spaceflight experiments. Seven or fourteen days of weightlessness in Russian biosatellites caused acceleration of lens and limb regeneration by an increase in cell proliferation, diffemntiation, and rate of morphogenesis in comparison with ground controls. After a comparable time of clinorotation the results obtained with T~~~~ ~lgar~~ using a horizontal clinostat were similar to those found in spaceflight. In contrast, in Pleurodeles wdtl using both horizontal and radial clinostats the results were contradictionary compared to Triturus. We speculate that different levels of gravity or/and species specific thresholds for gravitational sensitivity could be responsible for these contradictionary results.
The optimal answer to an injury in living systems is perfect regeneration of the missing parts. in most vertebrates posttraumatic regeneration capacity is negligible. However, in urodeles ~~rn~~i~j~}, regenerative potencies are not restricted to physiologic mg~em~on and wonnd healing only, but also involve the capability to develop perfect organ and body parts similar to the ontogenetieally formed originals. Eye, limb, and tail are those body parts which best form externally visible regenerates. These systems are very well described 111,/2/. Regeneration experiments in space were first proposed in 1984 /3/,/4/, and first carried out in 1985 aboard a Russian BIOCOSMOS satellite followed by six further spaceflights focusing on regeneration during the last decade. It was shown that when lenses were removed and limbs were amputated, ~gene~on was generally compile in both sp~~i~t weightlessness and lg ground controls. However, in all expe~men~, space-related factors provided specific stimulative effects, such as acceleration in regeneration rate and differentiation /5/, /6t, 171,/Si, 19/. Moreover, for eye regeneration, a remarkable enhancement of size of lens regenerates was found after spaceflights of animals whose lenses had been removed one week before start 151, 161, 171, 181. Addition~ly, abno~~ shares of the newly formed lenses (e.g., cataracts) and of regenerated forelimbs (e.g., skeleton malformation) were also observed, possibly related to other spaceflight factors than weightlessness /5/, 161,i7l. By advancing the animals to various regeneration stages before start, it was possible to determine the most sensitive stages of lens and limb regenemtion. It was shown that spaceflight effects occur mainly at early stages of lens and limb regeneration /6/. It was also observed that the influence of spaceflight on cell proliferation and differentiation has long-term character /9/. The spaceflight factors in~uen~ing regeneration are not only weightlessness, but also changes in light/dark cycle, temperature, low dose irradiation, vibration, and short term hypergravity during
H.J.htOBet al.
WY56
1~~~ and landing time. The infIuenee of such factors on mgene~on in urodeles as temperature flO/, fll/, /12/, season 1131, body size and age 1141, x-rays /15/, 1161,f171, 1181, /19/, and ligh~d~ cycle /20/,/21/,/22/, was described earlier under ground laboratory conditions. In order to better understand the specific influence of weightlessness on regeneration, all known or expected changes in Ytiors taking place in space were simulated as best as possible parallel to each spaceflight in ground control experiments /5/, lb/, /‘J/, ISI, 191. Among the different means to study the effect of gravity alterations, the effect of fast clinoro~on was used to investigate the influence of novel gravitational fields on different biological models such as cell cultures, developing animal embryos, and plants /23/, /W, 1251. To investigate the influence of low gnwity on adult urodeles, the main objectives of the present work were: 1) to develop adequate conditions to simulate microgravity in experiments with urodeles, 2) to find the effects of sirn~~ micro-g on regeneration in urodeles and 3) to compare the results with those which have been found in space experiments aboard the BIOCOSMOS satellites. MATERIALS AND METHODS Instruments and Conditions Two types of fast rotating clinostats were used. The first type was equipped with two ‘horizontally-oriented’ cuvettes ~O~~no~t~ (fig. 1, left) in which the rotation axis run through the cuvette center. Newt’s cranio-caudal axis was aligned as well as possible in the center, parallel to the rotation axis. Speed of cuvettes was 60 rotations per minute according to calculations showing that at this speed low g levels, i.e., mostly microgravity is reached. Humidity-satumtion of surrounding was achieved by placing a dish, approx. 300 cntz, filled with distilled water, on the bottom of the clinostat box. During rotation the ~~~d~ cycle was 0124 h; however7 5 min of lighting per day were allowed to check the animal’s state and the exposure conditions. The temperature was 2&l°C. The second type of clinostat (fig. 1, right) had six radially-oriented plates with horizontally oriented rotation axis (RO- clinostat). The correct position of the newts on the plates was reached by putting them into an elastic knit cotton mantle (Stiilpa/Hsrtmamr) fixed by clamps at the edge of the plates. Iu this way the cranio-caudal axis of the animals coincided with plate radius, but regenerating forelimbs and eyes were located in the center of rotation. The bottom of the clinostat box was filled with tap water approx. 2 cm in depth. The rotating plates had steady contact with water at their periphery, and the ends of the cotton mantles dipped in it during each rotation. This guaranteed 100% humidity inside the clinostat box, and moist surface of animals, thereby protecting the skin from drying. Plate speed, temperature, and the light/dark cycle were the same as in the HO- &no&t. Fig. l.(lef?) horizontally oriented (HO-) clinostat (Briegleb) and (right) radially oriented (RO-) clinostat (Anton) In the experimental controls, the same conditions were maintained, except clinorotation (fig. 1) and resembled those aboard the biosatellites, In the HO- clinostat the maximum possible distance of any body parts from the rotation center ranged from 5 to 8 mm for Pleurodeles and from 1 to 2 mm for ?“turus. In the RO- clinostat, the head and forelimbs were not farther from center than in the HO- clinostat, but for the other body parts, e. g., hind limbs and tails there was exposure to increasing gravity vectors. The HOclinostat was preferred, because it provided much less mechanical noise than the OR-clinostat. Animals Adult ~~~~rodel~~ ~a~~~(total length 100 to 110 mm and weight 5 to 6 g respectively, bred at the fnst. of Dev. Viol. Moscow) and T~~~~ vulgaris (t&al length SO-55 mm and weight lg approx., bred at
clinostat Influenceon Organ Regeneration
16i7~57
Zool. Inst., Univ. of Cologne) were used in our experiments. The latter was used for the most part because of its low size and weight, Experimental procedures ln accordance with the procedures of the spaceflight experiments f5f, 161,/7f, 181,the animals underwent operation 5-7 days before clinorotation. After anesthesia (MS 222 1:2000), lenses were microsurgically extirpated from both eyes through a small comeal incision, both forelimbs were amputated at the proximal ~ugopo~~ (forearm) level, and tail halfway between cloaca and tail tip, i.e., regeneration always took place simultaneously in three different body parts under ‘multiple lesion conditions’. Experimental and control animals underwent operation at the same time and were kept before clinorotation under similar conditions (21& lo C, light/dark cycle 12/12 h). The experimental animals were put in place in both HO- and OR-clinostats just before the beginning of the rotation period of 7 or 14 days. During this time the animals were not fed (to correspond with the space flight conditions).Upon removal from the clinostats, all animals were healthy. As a behavioral response to change of ‘gravity field’, they moved backwards for some minutes. To investigate the cell proliferation rate, some experimental and control animals were injected ~~e~~ne~ly with 3H-thymidine immediately after clinorotation and several others one week later (5lKi/g body weight, ‘Isotope’, Moscow). After four hours of incubation, the animals were anesthetized, sacrificed and samples (eyes, limbs, and tail) were fixed in Bouin’s fixative solution. From the remaining animals, the fully regenerated lenses were reextirpated and forelimbs and tails reamputated. The following second regeneration period was also observed. The regenerative progress was ascertained by determining the stages of lens /26/ and forelimb regenerates and by measuring the size of lens and tail regenerates /27/,/28/. For skeleton analysis, forelimb regenerates were stained ‘in toto’ with Vista-blue. Histologic~ and autor~og~hic~ analyses were performed in accordance with accepted methodsi51. RESULTS Lens regeneration in newts Tritmwsexposed in HO- clinostat for 14 days No notable differences in proliferative activity and stages of the lens regenerates were found at starting time: cell proliferation in the imrer layer of dorsal iris (source of lens regeneration) was initiated CH labeling index = 3 to 5%) and the regenerates achieved stages I-II both in experimental and in control animals. Just after the end of the rotation period, regenerates in experimental and control groups were advanced and showed non-synchronous stages IV-VIII. However, in comparable stages, the proliferation rate of iris cells was 1.3 - 1.5 times higher than in the controls (fig. 2a). This increase was also observed in tissues not involved in lens fo~~on (e.g., epide~~ cells of the dorsal eye lid). Ad~tion~ly, in one of 20 eyes of the clinorotated animals, a voluminous area of pigmented epithelial cells had developed, instead of a lens regenerate. Histologically, this area was characterized by numerous mitotic figures and the majority of cell nuclei had increased in volume (S-, G2-phase). Obviously, a melanoma had developed (fig. 3). A rn~i~~t growth of this type occurs very seldom in newts and was not found in any of our control and space flight experiments with Pleurodeies. The loss of proliferation control was possibly associated with the induced increase of cell proliferation.
orig con exp
con exp
b Fig. 2. ;a) 3H-Td labeling index of lens regenerates at start (stage 11)and 14 days’later at end (stage VII) of clinorotation; (b) mean diameter of original, control and clinorotated lens regenerates 1. reg. = one month, 2. reg. = two and half months after ending rotation, AU = arbitrary units; (c) projection of lens regenerates (mean volumes);
b
a
C
e d Fig, 3. Lens regeneration in Trifurus. Top: whole eyes; (a) control; (b) experimental animal. Note the opaque anterior eye chamber; (c) e~e~rnen~ animal with melanoma fo~ation. Note the prodding cornea. Bottom: histology; (d) median section of the right eye. Note the connection of the tumor with the dorsal iris margin; (e) higher m~nifi~ion. C = cornea, NR = nenral retina, RP = retinal pigment epithelium, T = tumor, I = upper and lover iris margin, p = pigment gran~es, t = mitotic figures
Fig. 4. Extirpated lenses (Trifzms) (a) original lens, @) newly formed control lens, (c) newly formed experimental lens. a
b
Fig. 5. Forelimb regenerates (a, b) just after clinorotation (c, d) three weeks later. ‘lbe experimental regenerates (b, d) are more advanced than the corresponding control regenerates (a, c). a
d
b
Fig. 6. An~po~~ skeleton rn~fo~~ons in limb regenerates of clinorotated animals (a) normal, Co), (c), (d) different grades of skeleton malformations. a
b
d
Clinostat lofluence cm Organ Regeneration
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Two months after clinorotation, the fully differentiated first lens regenerates were reextirpated and a second regeneration was initiated. Tbe mean lens diameter of the first experimental regenerates was approx. 110% the volme of that of the controls. The second lens regenerates, extirpated and measured 6 weeks after the first reextirpation, showed comparable differences in size (fig. 2b, c; 4). Forelimb regeneration in Triturus exposed in HO-clinostat for 14 days Forelimbs were amputated seven days before start of clinorotation. At start, wound healing had finished and the epidermal cap was formed in experimental and control groups. At end of clinorotation, the forelimb regenerates of rotated animals were markedly advanced, having reached the stages ‘cone’ to ‘palette’, whereas at the same time, control regenerates achieved stages ‘bud’to ‘cone’ only. Some typical differences between experimental and control regenerates are shown in figure 5, The proliferation rate t3HTd-labeling index) of the blastema cells of the experimental regenerates of corresponding stages ‘late bud’ and ‘early cone’ had advanced up to 1.4 and 1.3 fold, respectively, than those of the controls. Growth and morphological differentiation of the rotated regenerates were accelerated up to the ‘late 3 digit’ stage (fig. 7a).
a
b
regeneration stages
regeneration stages
days after landing
weeks after reamputation
Fig. 7. (a) first and fb) second forelimb ~genemtion of ~r~~~r~svdgaris at& 14 days of clinorotation; C = control, E = clinorotated limbs, d = days, w = weeks, % = percentages of stages at different time.
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Two months after ending rotation, the regenerates were reamputated and the following second regeneration was screened again; it showed that acceleration in the experimental group still continued for up to three and half months (fig. 7b). In contrast with the results obtained in the spa&light experiments, in which an increased number of skeleton malformations among the regenemted forelimbs was observed, the limb regenerates of the clinorotated animals showed no notable differences in frequency and range of abnormalities (see table and fig. 6a, b, c, d).
Table: Skeleton malformation in Triturus forelimb regenerates. group are statistically not significant.
Differences
in experimental
and control
Tail regeneration in Triiurus exposed in HO-clinostat for 14 days Similar to forelimb regeneration, the tail regeneration rate in the clinorotated newts was also higher at all time points. Just before start, the tail stumps of all animals were in the same stage: the wound was closed, an epidermal cap had formed and under it dedifferentiation of dermis, musculature, vertebra, and spinal cord had set in. After ending rotation, the tail regenerates were essentially advanced and reached stages IV - V in the experimental group, in contrast in the control group stages III-IV. In order to allow the tails to grow further, cell proliferation rate was not analyzed at these stages. But some weeks later the absolute length of the regenerated tails and the ratio of regenerate/whole tail length demonstrated the higher growth rate in the experimental series (fig. Sa, b). 100
a Fig. 8. Tail regeneration
con
exp
b
con
exp
in Trim-us (a) tail length (arbitrary units), (b) length ratio (reg./whole tail)
Regeneration in Pleurodeles exposed in HO- and RO- clinostat As a consequence of the small size of Tritums, its position in the HO-clinostat was very close to the rotation axis: the longest distance of animals periphery from center was 1 to 2 mm, so that calculated g ranged between 4.0x10-3 to 8.0~10~~ g (fig. 9a). On this basis, we assume that this experimental series provided a good parallel with real space flight conditions in the Biocosmos satellites. Since spaceflight experiments have been carried out with Pleurodeles, we were also interested in studying the influence of clinorotation on regeneration in this species, especially due to the larger size of the animal, the simulated low gravity was higher than for Triturus. Additionally, in the RO-clinostat the animals were ‘radially-oriented’, so that only the area where regeneration takes place was at the rotation center.
0,30 ________:___-____;;,~--_I_ .~~~~ 'g 0,20 -- -_____:;;~__~___.i___ 2 012
3
4
5 6
7
8
910 mm
distance from center
0,lO - - _,,_d'_____________-___ 012
3
4
5
6
7
distance from cemz
8
910
cm
b a Fig. 9. Changes of calculated gravitational fields relative to the rotated animals: (a) HO- clinostat; (b) RO- clinostat. Lens, forelimb, and tail regeneration in Pleurodeles exposed in HO- clinostat for 14 days The distance of the regenerating parts of PZeurodeles from rotation axis rauged between 5 and 10 mm; i.e., the calculated g-forces varied from 3.2x10-2 to 4.O~lO-~g( fig. 9a). It was surprising that under these low gravity conditions lens, forelimb, and tail regeneration rate was reduced. Before start of clinorotation, histological analysis of all lens regenerates showed stages I to II. After rotation, lens regenerates had achieved stages III to V and the ~s~b~on was relatively similar in both groups. However, 10 days later differences between experimental and control groups were notable: progress in regeneration of the clinorotated animals was reduced and showed, stages III to VII, more divergence than the control regenerates, which during the same period had advanced to stages VII to IX. In forelimb and tail regeneration, analyzed by the same methods as for Triturus, the rotated Pleurodeles showed a comparable reduction as seen in lens regeneration. Lens and forelimb in Pleurodeles exposed in RO- clinostat for 7 days Since in the RO-clinostat only the upper part of the newt’s body was exposed in the area of low gravity simulation, only lens and forelimb regeneration was investigated (tails were not amputated). In spite of this, the peculiarities found in lens and limb regeneration reflected those observed in the experiment with PZe~rodeles rotated in HO- clinostat; they suggest retarded lens and forelimb ~gene~on as well. It should be emphasized that only head and forelimbs were influenced by low gravity forces {approx. 10m2g).The other parts were exposed to conthmously increasing gravity (up to approx. 0.4g ) {fig. 9b). DISCUSSION AND FUTURE ACTIVlTIES The results of our investigations show a clear effect of simulated weightlessness on regeneration in urodeles, in particular, on cell proliferation, tissue growth, and organ regeneration rate. In all series of experiments we found clear, lasting responses which were similar in all studied organs (eye, limb, tail), but varied in dependence on the level or quality of simulated gravity reduction and animal species. The retarded cell p~life~on and regeneration rate in Ple~rode~e~ under conditions of clinoro~on was unexpected, given the results obtained in all space fight experiments (/5/,/6/~7/,/8~. However, the lowest level of gravity that we achieved under the conditions employed was approx. 10-z g. Thus, the ‘slowing down’ of regeneration of the rotated Pleurodeles may have been induced by this. There is no information in literature on the effect on multicellular organisms of long-term exposure to gravitational fields between less than lg and far above ‘micro-g (10-6g). Our data may be the first of this kind. However, we should not discount other factors which could possibly induce the described retardation, e. g., mechanical ‘noises’ produced by rotation of the relatively big and heavy animals (especially in the case of RO-clinostat). In contrast to Pleurodeles, the calculated gravity in the experiments with Triturus was lower. It reached IO3 to 10m4g(HO-clinostat). Precisely these g values were suspected to be a possible threshold of sensitivity of biological processes to acceleration 1241. So, it is reasonable to assume that Trj~~~ rotated 14 days in HO-clinostat lived under ‘sub~~shold acceleration’ or ‘funotional weightlessness’ conditions /29/, 1301. As Briegleb 1241 pointed out, most sensitive acceleration processes may have a threshold far above 10m4gand evidently there are thresholds near lg or above it. However, in the absence of any quantitative data on the effect of acceleration on different
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animals, whether from our own experiments or from literature, we can specuiate that the predominantly water-living Pleurodeles and the predominantly land-living Trifurus could express difibrent ‘sensitivities’ to acceleration. Our results support the hypothesis that moderate alone weigh~essness stimulates regeneration, whereas low-gravity fields higher than 1Q3g could have an inhibitory effect on regeneration. It is also possible that g vectors higher than 10e3gdo not have any effect at all and that ‘big’ newts, Pleurodeles, are more sensitive to mechanical noises (e.g., vibration) produced by the rotation, From experiments with bacteria in Dl and D2 spaceflight missions, it is known that different culture media can be responsible for contradictory results, a phenomenon that has been confirmed by clinostat e~e~rnen~ 13II. The effect of fimctional weightlessness was independent of other environmental conditions. More rapid cell proliferation and tissue growth during regeneration in Trifurus under the conditions observed in spaceflights (BIOCOSMOS satellites), were actually caused by weightlessness, and not by other spacerelated factors. Acceleration of cell m~tipli~on under simulated and real ‘micro-g’ represents our main finding, it is a crucial event in the regenerative processes, and the prerequisite for subsequent differentiation and morphogenesis, which are strongly coordinated and programmed. From current knowledge, cell proliferation in animals is controlled by mechanisms and factors, e. g., well determined growth factors (cytokines), cell microenvironment, cell shape changes, cell to cell and cell to substrate contacts, individual cell gene expression. Our experiments have demonstrated that the mechanisms which regulate cell proliferation under normal gravity, also work under novel gravitational conditions: They also suggest that the basic mechanisms of cell control such as receptor binding and signal transduction are not, in principal, altered by gravity. In experiments in which the effect of simulated ‘micro’- and ‘hypergravity’ on different cell types ‘in vitro’ were studied, it was shown that moderate novel gravity has an effect at particular steps in the signal transduction cascade, resulting in altered patterns of gene expression and ultimately in DNA replication and cell division 129, /32/, /33/. As a result it was suggested that pm~fe~on of cells cultured in a h~ergm~~on~ field could be increased /341, but under microgravity conditions it is markedly decreased 1351. This suggestion is based on data obtained for normal and malignant cells, mostly mammalian, ‘in vitro’, and points to a direct influence of gravity changes on cell proliferation regulatory mechanisms. This is why our data obtained in (multicellular) adult vertebrates (newts) exposed to simulated weightlessness and data obtained on cell cultures cannot really be compared. Moreover, we believe that in our case the influence of wei~~es~ess, either simulated or in space, is first mediated by low-g reactivity of the whole organism. This reactivity, that could be named ‘microgmvitational stress’ is still far from being completely understood, Our work presents evidence on the regulatory role of common integrating systems in organisms, one of them being long-term augmentation of lens, forelimb and tail regeneration rate, after relatively short (7 to 14 days) exposure to real and simulated weightlessness: 1.) three months after return to normal 1 g conditions, the higher proliferation and differentiation rate continued, demonstrated by the first and second ~gene~on; 2.) in Trials an increased mgene~ion rate was found in all regenerating systems analyzed (eye, limb, tail); 3.) the increased cell proliferative activity determined in epidermis (eye lids) which was not involved in eye regeneration. From our results, the hypothesis that gravitational stress is nonspecific becomes improbable. But we do not want to definitively reject this idea, since it seems reasonable to assume that rapid rotation of animals can produce stress factors. We have shown that the reaction of cells to wei~~essness is longterm. However, reversibility has not been shown to date. It could be that when normal gravity is ‘switched off, cells forget or lose the mechanism which was a prerequisite for survival at the beginning of evolution. This mechanism never needed to be protected until the beginning of the spaceflight era. Because of this, there was no necessity to develop a kind of ‘switch on’. The energy freed by the switch off process could be directed to accelerate the regeneration process. There is one indication from our results that cell prolife~on under normal lg conditions is less likely to degenerate than under conditions of weightlessness: the single case of melanoma formation (a very rare event in newts), which we found in the experimental group of 10 animals with 20 lens regenerates. As an important result of our experiments, we have shown that small, lower vertebrates, especially land-living animals such as Tribes are suitable for experiments under real and clino~t-sim~ated
ciinostal Iofluence 081 Organ Regeoeration
WW
spaeeflight conditions, much better than cell or tissue cultnres. The advantages are: 1.) the living system itself, which guarantees stable conditions during the whole experiment; 2.) artificially induced processes which cause natural reactions with no need for artificial support; 3.) within certain limits the animals need no food aud special care to survive and regenerate (one month and more); 4.) independence of animal survival from fluctuation of temperature in limits of 0 to +28’C (poikilothemral animals); 5.) multiple surgery and regeneration (lens, tail, limb, gu& brain, spinal cord, peripheral nerves) without excessive stress. 6.) the animals are suitable as a system of cell and tissue cultures, i.e., carrying transplants in body cavity, eye chamber etc. Given these advantages, many questions concerning the influence of novel gravitational fields can be tested in clinostat experiments.
1. R.J. Goss, Principles of Regeneration. Academic Press, Inc. London (1969). 2. H. Wallace, Vertebrate limb regeneration. John Willey and Sons, Chichester (198 1) 3. H.J. Anton, Die Extremitatenregeneration der Amphibien, ein geeignetes Modell zur Untersuchung evolutorischer und modifikatorischer Wirkung der Schwerelosigkeit. LXVLR-Mtt. 1985,63- 68 (1985). 4. H.J. Anton, Limb regeneration in ~phibi~s a suitable model for the inve~g~on of evolutions and rno~~~~~ effect of weigh~es~ess, European Space Agency TT @A T)T 1987,47-5 1 f 1988). 5. V.I. Mitashov, E.N. Grigoryan, S.Y. Tuchkova and E.M. Cherdanzeva, Organs and tissue regeneration in Amphibia under the space flight conditions. In: Life science research in Space. DA and ESTEC Publ., -I 1987 Netherlands, 299-303 (1987). 6. V.I. Mitashov, E.N. Grigoryan , S.Y. Tuchkova, E.A. Oigenblick and I.E. Malchevskaya, Lens and limb regeneration in the newt during and after 13 day long spa&light. ESA and ESTEC Pub1 1990. Netherlands, 85-92 (1990) 7. V.I. Mitashov, E.N. agony, S.Y. Tuchkova, E.A. Oigenb~ck, I.E. M~chevskay~ Lens and limb regeneration in Amphibia under the spaceflight conditions. In: Recent Trends in Regeneration Research. (V.Kiortsis, SKoussoulakos, II. Wallace eds.), Plenum Pubi. Corp., New-York, 135-136 (1989). 8. E.N. Grigoryan, E.A. Oigenblick, SYa. Tuchkova, V.I. Mitashov, Results of investigations aboard Russian biosatellites. Nuuka m,345-350 (in Russian) (1992). 9. N.V. Bruchlinskaya , S.Y. Tuchkova , E.N. Grigoryan , H.J. Anton and V.I. Mitashov, Specialties of the influence of spaceflight related factors on regeneration processes in mammals and urodeles. Bulletin of Russian Acad.Sci., ser. Biol. (in Russian) (submitted) (1994). 10.0. N&unum, Temperature influence on lens ~g~e~on Proc.Imp.Acad.To~o. II, 121-124 (1935).
in urodele Triturus pyrrhogaster (Boie).
11. D.J. Donaldson aud F. Chau, The influence of temperature on lens regeneration in the aduh newt, Triturus viridescens. Growth, 37.69-76 (1973). 12. M.K. Schauble and M.Nentwig, Temperature and prolactin as control factors in newt forelimb regeneration. Jfip.2001. M,335-344 (1974). 13. M.K. Shauble, Seasonal variation of newt forelimb regeneration tmder controlled enviromnental conditions. ~~p.2001. 181.281-286 (1972). 14. R.W. Reyer, Regeneration of the lens in the amphibian eye. In “Regeneration” (D. Rudnick ed.), Ronald Press New-York, 21 l-265 (1962).
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15. V .V. Bnmst, The efl%ts ofxadiatioa on the eye .EXJX Eye Iies 2, f.16-122 (1966).
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17. S.Y. Tu~~ov~ Tissue resources active in restoring x-my depressed limb regenerative capacity in Axolotl..&wcee~ ~~~cff~ S’ei.176,1181-1184 (1967). 18.3X. U~~~ller, The actiou of x-~i~~u on the regene~on new& Diemictylw viridescens. ~~~.Zoo~. 168,403-421(1968).
field of the forelimb of the adult
19. M. Mada, H. Wallace, How x-rays in&bit amphibian limb mge~~~~~. JEscp. Zool., 197,105-I 13 (1976). 20. R.A. Borsuk, V.V. Popov, ~fluen~ Proceed. USS’RAcad. Ski., 9,
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21. F. Ghan, Comparison of lens mgeue~~~ in adnlt newts, Tritums viridescens, between light and dark prcsdqmd and non pact animals, A~a~.R~~.~21fz. 521-525 (1982). 22. C.E. Maier, M. Singer, The e&et. of limiting light to the pineal on the rate of forelimb mge~~o~
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23. A. Cog&, FK. Gem~der, Gravity e&c& on single cells: ~c~iq~~~~ ~~n~~ Advances in $$p.xe biology and medicine, 1, 177-241 (199 1).
24. W. Kegleb, Some qu~i~ve and quantitative aspects on the f&t- ra~~g tool, ASGS’B- BulIefin, 2 23-30 (1992).
and theory.
diuostat as a research
25. R.P. De Grcrot, P.3, Rijken, 1. den Iiertog, J, Boonstra, A.J. Verldeij, S.W. de La&, W. Kmijer, %&ear responses to protein k&se C signal ~du~on are sensitive to gravity changes. .&$a~~~~~~~,, .@7,87-90 (1991).
27. L.E. Iten> S.V. Bryant, Forelimb regeneration from different levels of amputation in the newt ~o~ph~~rnus viridescens: length, rate and stages. R~~‘Arc~jv~r Entw. me&. Org., 173.263-282 (1973).
29. HJ. Anton, EN Grigoryan, Sim&ted miaogmvity induces increase of regeneration rate, cell proliferative. activity, and e~~~rn~t of size of regenerate during Wolffian lens regeneration in the newt. 86. i/&h. ~eu~~c~e Zvol. Ges I993 ~~~ur~ ~~~~~~~~~~~en~~ j&, 204 (1933). 30. I-U. Anton, EN. Grigoryan, Altered in&ewe of gravity can provide long-term effect on forelimb and tail regeneration in the newt. Cpreliminwy results of experiments on simulated microgravity) 86. K&z. ~~f~c~~ Zoof. Ges. I993 ~~~~~~g ~~~~&~j~~~~~~, fi 203 {1993).
ClinostatIniluenee on Organ Regeneration
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