Effect of hypergravity on carboanhydrase reactivity in inner ear ionocytes of developing cichlid fish.

It has been shown earlier that hypergravity slows down inner ear otolith growth in developing fish. Otolith growth in terms of mineralization mainly depends on the enzyme carboanhydrase (CA), which is responsible for the provision of the pH-value necessary for calcium carbonate deposition. Larval siblings of cichlid fish (Oreochromis mossambicus) were subjected to hypergravity (3 g, hg; 6 h) during development and separated into normally and kinetotically swimming individuals following the transfer to 1 g (i.e., stopping the centrifuge; kinetotically behaving fish performed spinning movements). Subsequently, CA was histochemically demonstrated in inner ear ionocytes (cells involved in the endolymphatic ion exchange) and enzyme reactivity was determined densitometrically. It was found that both the total macular CA-reactivity as well as the difference in reactivities between the left and the right maculae (asymmetry) were significantly lower (1) in experimental animals as compared to the 1 g controls and (2) in normally swimming hg-animals as compared to the kinetotically behaving hg-fish. The results are in complete agreement with earlier studies, according to which hypergravity induces a decrease of otolith growth and the otolithic calcium incorporation (visualized using the calcium-tracer alizarin complexone) of kinetotically swimming hg-fish was higher as compared to normally behaving hyper-g animals. The present study thus strongly supports the concept that a regulatory mechanism, which adjusts otolith size and asymmetry as well as otolithic calcium carbonate incorporation towards the gravity vector, acts via activation/deactivation of macular CA.     

Influence of hypergravity on fish inner ear otoliths: I. Developmental growth profile.

Inner ear stones (otoliths) of larval cichlid fish Oreochromis mossambicus were marked with the calcium-tracer alizarin-complexone (AC) at 1 g earth gravity before and after a 3, 7, 14 or 21 days stay of the animals at hypergravity conditions (hg; 3 g, centrifuge). After the experiment, the otoliths' area between the two AC-labellings was measured with regard to size and asymmetry (size difference between the left and the right stones). Both utricular and saccular otoliths (lapilli and sagittae, respectively) continued growing in a linear way at hg, but growth was significantly slowed down as compared to parallely raised 1 g-control specimens. In case of bilateral asymmetry between the corresponding otoliths its formation in hg-animals became reduced as compared to the 1 g controls. The reduction of asymmetry was much more pronounced in the sagittae than in the lapilli. The latter result supports an earlier hypothesis, according to which especially a low sagittal asymmetry has a functional advantage. In general, the results strongly suggest that otolith growth is continuously regulated in dependence of the environmental gravity vector.     

Influence of hypergravity on fish inner ear otoliths: II. Incorporation of calcium and kinetotic behaviour.

Larval siblings of cichlid fish (Oreochromis mossambicus) were subjected to hypergravity (hg; 3 g, 14 days) during development. Following the transfer to 1 g (i.e., stopping the centrifuge) they were separated into normally and kinetotically swimming individuals (the latter performed spinning movements). During hg, the animals were maintained in aquarium water containing alizarin-complexone (AC), a fluorescent calcium tracer. Densitometric measurements of AC uptake into inner ear otoliths (optical density of AC/micrometers2) revealed that the kinetotic individuals had incorporated significantly more AC/calcium than the normally behaving fish. Since the amount of otolithic calcium can be taken as an approximation for otolith weight, the present results indicate that the otoliths of kinetotically swimming samples were heavier than those of the normally behaving larvae, thus exhibiting a higher absolute weight asymmetry of the otoliths between the right vs. the left side of the body. This supports an earlier concept according to which otolith (or statolith) asymmetry is the cause for kinetoses such as human static space sickness.     

Ultrastructural aspects of otoliths and sensory epithelia of fish inner ear exposed to hypergravity.

The present electron microscopical investigations were directed to the question, whether alterations in the gravitational force might induce structural changes in the morphology of otoliths or/and inner ear sensory epithelia of developing and adult swordtail fish (Xiphophorus helleri) that had been kept either under long-term moderate hypergravity (8 days; 3g) or under short-time extreme hypergravity (10 minutes up to 9g). The otoliths of adult and neonate swordtail fish were investigated by means of scanning electron microscopy (SEM). Macular epithelia of adult fish were examined both by SEM and transmission electron microscopy (TEM). The saccular otoliths (sagittae) of normally hatched adult fish revealed an enormous inter- (and even intra-; i.e. left vs. right) individual diversity in shape and size, whereas the otoliths of utricles (lapilli) and lagenae (asterisci) seemed to be more constant regarding morphological parameters. The structural diversity of juvenile otoliths was found to be less prominent as compared to the adults, differing from the latter regarding their peculiar crystalline morphology. Qualitative differences in the fine structure (SEM) of otoliths taken from adult and larval animals kept under 3g in comparison to 1g controls could not be observed. The SEM and TEM investigations of sensory epithelia also did not reveal any effects due to 3g stimulation. Even extreme hypergravity (more than 7g) for 10 minutes did not result in distinct pathological changes.     

Size and cell number of the utricle in kinetotically swimming fish: a parabolic aircraft flight study.

Humans taking part in parabolic aircraft flights (PAFs) may suffer from space motion sickness (SMS, a kinetosis). Since it has been repeatedly shown earlier that some fish of a given batch also reveal a kinetotic behavior during PAFs (especially so-called spinning movements and looping responses) and due to the homology of the vestibular apparatus among all vertebrates, fish can be used as model systems to investigate the origin of susceptibility to motion sickness. Therefore, we examined the utricular maculae (they are responsible for the internalization of gravity in teleosteans) of fish swimming kinetotically at microgravity in comparison with animals from the same batch who swam normally. On the histological level, it was found that the total number of both sensory and supporting cells of the utricular maculae did not differ between kinetotic animals as compared to normally swimming fish. Cell density (sensory and supporting cells/100 micrometers2), however, was reduced in kinetotic animals (p<0.0001), which seemed to be due to malformed epithelial cells (increase in cell size) of the kinetotic specimens. Susceptibility to kinetoses may therefore originate in malformed sensory epithelia.     

Effects of heavy ion radiation on the brain vascular system and embryonic development.

Using neonatal rats as a model system, we investigated the response of the brain vascular system to ionizing radiation and found that distinct petechial hemorrhages developed in the cerebral cortex within a few hours after irradiation, reached a maximum about 13 to 24 hours, and decreased exponentially with time. No brain hemorrhage was found in neonatal rats 12 days after irradiation. Our experimental results indicate that a dose of a few hundred rad of X rays can induce a significant number of hemorrhages in the brain, and the number of lesions increases exponentially with dose. Heavy ions induce more hemorrhages than X rays for a given dose, and the RBE for 670 MeV/u neon particles ranges from about 2.0 for low doses to about 1.4 for high doses. A histological study of the hemorrhages indicates that a large number of red blood cells leak from the blood vessels. The radiation-induced hemorrhages may be a result of some capillary membrane damages or reproductive death of some blood vessel epithelial cells. The fast onset of hemorrhage after irradiation suggest that some membrane damage may be involved. The effect of heavy-ion radiation on the embryonic development was studied with energetic iron particles. Pregnant mice were whole-body irradiated with 600 MeV/u iron particles on day 6 of gestation and were sacrificed 12 days after irradiation. Various physical abnormalities were observed, and embryos irradiated with 1 rad iron particles showed retardation of body development.     

The white blood cell line: changes induced in mice by hypergravity.

The effect of hypergravity on the white blood cell (WBC) line of mice was investigated by use of horizontal centrifuge. Several sets of experiments were performed, in which the parameters measured were the WBC and differential cell count in the peripheral blood. In another experiment, lymphocyte counts from the spleen, lymph nodes, and the thymus were measured. The needed samples were taken from the mice during a stay of 7-40 days under a hypergravity of 1.6G. The test groups that were placed on the arms of the centrifuge (1.6G) were compared with stationary control groups (1G) and a rotating control group located at the center of the centrifuge (1G). Such a comparison revealed the test animals to be deficient on all counts, to wit, showing a decrease in total number of WBC's, a decrease in lymphocyte number in the peripheral blood and a decrease in the number of lymphocyte in the spleen and thymus. The decrease of lymphocytes in peripheral blood was characterized by two different slopes--an early and temporary decrease at the first days of the experiment evident in both test and rotating control groups followed by a temporary increase, and a later persistent decrease, evident only in the test group, while in the rotating control lymphocyte counts reverted to normal. There were no significant differences in monocyte or neutrophil counts, except for a temporary increase in the number of neutrophils which peaked on the seventh day. In order to evaluate the effect of hypergravity on restoration of hematopoiesis following hematopoietic suppression, 5-fluoro-uracil (5-FU) was administered i.v. to both the experimental and control mice. Suppression of bone marrow was observed in all groups injected with 5-FU, but while there was later an increase in cell counts in the control groups, there was no such increase in the test group subjected to hypergravity.     

Effects of altered gravity on the swimming behaviour of fish.

Humans taking part in parabolic aircraft flights (PAFs) may suffer from space motion sickness-phenomena (SMS, a kinetosis). It has been argued that SMS during PAFs might not be based on microgravity alone but rather on changing accelerations from 0 g to 2 g. We test here the hypothesis that PAF-induced kinetosis is based on asymmetric statoliths (i.e., differently weighed statoliths on the right and the left side of the head), with asymmetric inputs to the brain being disclosed at microgravity. Since fish frequently reveal kinetotic behaviour during PAFs (especially so-called spinning movements and looping responses), we investigated (1) whether or not kinetotically swimming fish at microgravity would have a pronounced inner ear otolith asymmetry and (2) whether or not slow translational and continuously changing linear (vertical) acceleration on ground induced kinetosis. These latter accelerations were applied using a specially developed parabel-animal-container (PAC) to stimulate the cupular organs. The results suggest that the fish tested on ground can counter changing accelerations successfully without revealing kinetotic swimming patterns. Kinetosis could only be induced by PAFs. This finding suggests that it is indeed microgravity rather than changing accelerations, which induces kinetosis. Moreover, we demonstrate that fish swimming kinetotically during PAFs correlates with a higher otolith asymmetry in comparison to normally behaving animals in PAFs.     

CNS development under altered gravity: cerebellar glial and neuronal protein expression in rat neonates exposed to hypergravity.

The future of space exploration depends on a solid understanding of the developmental process under microgravity, specifically in relation to the central nervous system (CNS). We have previously employed a hypergravity paradigm to assess the impact of altered gravity on the developing rat cerebellum. The present study addresses the molecular mechanisms involved in the cerebellar response to hypergravity. Specifically, the study focuses on the expression of selected glial and neuronal cerebellar proteins in rat neonates exposed to hypergravity (1.5 G) from embryonic day (E)11 to postnatal day (P)6 or P9 (the time of maximal cerebellar changes) comparing them against their expression in rat neonates developing under normal gravity. Proteins were analyzed by quantitative Western blots of cerebellar homogenates; RNA analysis was performed in the same samples using quantitative PCR. Densitometric analysis of Western blots suggested a reduction in glial (glial acidic protein, GFAP) and neuronal (neuronal cell adhesion molecule, NCAM-L1, synaptophysin) proteins, but the changes in individual cerebellar proteins in hypergravity-exposed neonates appeared both age- and gender-specific. RNA analysis suggested a reduction in GFAP and synaptophysin mRNAs on P6. These data suggest that exposure to hypergravity may interfere with the expression of selected cerebellar proteins. These changes in protein expression may be involved in mediating the effect of hypergravity on the developing rat cerebellum.     

Effects of gravity on early development.

The development of embryonic and larval stages of the South African Toad Xenopus laevis D, was investigated in hyper-g up to 5 g (centrifuge), in simulated 0 g (fast-rotating clinostat), in alternating low g, hyper-g (parabolic flights) and in microgravity (Spacelab missions D1, D-2). The selected developmental stages are assumed to be very sensitive to environmental stimuli. The results showed that the developmental reaction processes run normal also in environments different to 1 g and that aberrations in behavior and morphology normalize after return to 1 g. Development, differentiation, and morphology of the gravity perceiving parts of the vestibular system (macula-organs) had not been affected by exposure to different g-levels.     

Regeneration of guinea pig facial nerve: the effect of hypergravity.

Exposure to moderate hypergravity improves the regenerative capacity of sectioned guinea-pig facial nerve. The improvement in regeneration is tri-directional as follows: a) an average 1.7 fold increase in rate of regeneration in guinea pigs subjected to hypergravity; b) a 25% enhancement of facial muscle activity following the exposure to hypergravity; and c) improvement in the quality of regeneration from an esthetic standpoint. A good correlation was recorded between the histological structure of the severed nerve at the end of the regeneration and the clinical results.     

Neuronal feedback between brain and inner ear for growth of otoliths in fish.

Previous investigations revealed that fish inner ear otolith growth (concerning otolith size and calcium-incorporation) depends on the amplitude and the direction of gravity, suggesting the existence of a (negative) feedback mechanism. In search for the regulating unit, the vestibular nerve was unilaterally transected in neonate swordtail fish (Xiphophorus helleri) which were subsequently incubated in the calcium-tracer alizarin-complexone. Calcium incorporation ceased on the transected head sides, indicating that calcium uptake is neurally regulated.     

Behavioural changes in Paramecium and Didinium exposed to short-term microgravity and hypergravity.

The swimming behaviour of two ciliate species, Paramecium caudatum and Didinium nasutum was analyzed under microgravity and hypergravity. In Paramecium the differences between former upward and downward swimming rates disappeared under weightlessness. At microgravity the swimming rates equalled those of horizontally swimming cells at 1g. In contrast, the swimming rates of Didinium increased under microgravity conditions, being larger than horizontal swimming rates at 1g. These findings are in accordance with a hypothesis of gravireception in ciliates based on electrophysiological data, which considers the different topology of mechanoreceptor channels in theses species. The hypothesis received further support by data recorded under hypergravity conditions.     

Growth restoration in azuki bean and maize seedlings by removal of hypergravity stimuli.

Hypergravity stimuli, gravitational acceleration of more than 1 x g, decrease the growth rate of azuki bean epicotyls and maize coleoptiles and mesocotyls by decreasing the cell wall extensibility via an increase in the molecular mass of matrix polysaccharides. An increase in the pH in the apoplastic fluid is hypothesized to be involved in the processes of the increase in the molecular mass of matrix polysaccharides due to hypergravity. However, whether such physiological changes by hypergravity are induced by normal physiological responses or caused by physiological damages have not been elucidated. In the present study, we examined the effects of the removal of hypergravity stimuli on growth and the cell wall properties of azuki bean and maize seedlings to clarify whether the effects of hypergravity stimuli on growth and the cell wall properties are reversible or irreversible. When the seedlings grown under hypergravity conditions at 300 x g for several hours were transferred to 1 x g conditions, the growth rate of azuki bean epicotyls and maize coleoptiles and mesocotyls greatly increased within a few hours. The recovery of growth rate of these organs was accompanied by an immediate increase in the cell wall extensibility, a decrease in the molecular mass of matrix polysaccharides, and an increase in matrix polysaccharide-degrading activities. The apoplastic pH also decreased promptly upon the removal of hypergravity stimuli. These results suggest that plants regulate the growth rate of shoots reversibly in response to hypergravity stimuli by changing the cell wall properties, by which they adapt themselves to different gravity conditions. This study also revealed that changes in growth and the cell wall properties under hypergravity conditions could be recognized as normal physiological responses of plants. In addition, the results suggest that the effects of microgravity on plant growth and cell wall properties should be reversible and could disappear promptly when plants are transferred from microgravity to 1 x g. Therefore, plant materials should be fixed or frozen on orbit for detecting microgravity-induced changes in physiological parameters after recovering the materials to earth in space experiments.     

Fluxes of galactic iron nuclei and associated HZE secondaries, and resulting radiation doses, in the brain of an astronaut.

Although galactic iron nuclei constitute only a small percentage of the total flux of radiation in space, they are extremely significant from a biological standpoint, and represent a concern for long-term manned space missions of the future. Dosages resulting from iron nuclei, and the high-charge secondary nuclei subsequently produced in nuclear fragmentation reactions, have been calculated at the centre of a simple model of the human brain, shielded by various thicknesses of aluminium. Three mission scenarios are considered representing different geomagnetic shielding conditions at solar minimum. Without artificial shielding absorbed dose rates outside the magnetosphere, in polar orbit and in the proposed Space Station orbit, are approximately 0.3, 0.1 and 0.03 cGy/year respectively, corresponding to dose equivalent rates of 8.0, 2.5 and 0.8 cSv/year, and decreasing by roughly a factor of two behind 10 g/cm2 of aluminium. In line with new approaches to risk estimation based on particle fluence and track structure, calculations of the number of cell nuclei likely to be struck by these HZE particles are also presented. Behind 10 g/cm2 of aluminium, 3.4%, 1.3% and 0.5% of cell nuclei at the centre of the brain will be traversed at least once by such a particle within three years, for the three mission scenarios respectively.     

Effects of carbon ions on primary cultures of mouse brain cells.

Primary mixed cultures of astrocytes and microglia were obtained from neonatal mice, and were irradiated with high-LET carbon ions. Immunohistochemical staining showed astrocytes survived more prominently than microglia. Tagged with specific antibodies, astrocytes and microglia surviving after irradiation were counted by flow cytometry. Decreases in the number of microglia and astrocytes were detected at a dose as small as 2 Gy when Day 5 cultures were irradiated with 13 keV/micrometer carbon ions. When the cultures were irradiated on Day 10, the dose-dependent decrease of microglia was more prominent for 13 keV/micrometer carbon ions than 70 keV/micrometer carbon ions. Astrocytes showed a marginal decrease at Day 10 and Day 14. We concluded that microglia are more sensitive than astrocytes to carbon ions and X-rays, and that the radiosensitivity of microglia depends on both differentiation/proliferation status and radiation quality.     

Effects of gravity and cosmic rays on cell proliferation kinetics in Paramecium tetraurelia.

Space flights resulted in a stimulating effect on kinetics of proliferation in Paramecium tetraurelia. Additional experiments were performed in order to determine the origin of this phenomena. Paramecia were cultivated in balloon flights or in a slow clinostat, or were exposed to different levels of hypergravity. The results suggest that changes in cell proliferation rate are related to cosmic rays and to a direct effect of microgravity.     

Crickets in space: morphological, physiological and behavioral alterations induced by space flight and hypergravity.

"Crickets in Space" was a Neurolab experiment by which the balance between genetic programs and the gravitational environment for the development of a gravity sensitive neuronal system was studied. The model character of crickets was justified by their external gravity receptors, identified position-sensitive interneurons (PSI) and gravity-related compensatory head response, and by the specific relation of this behavior to neuronal arousal systems activated by locomotion. These advantages allowed to study the impact of modified gravity on cellular processes in a complex organism. Eggs, 1st, 4th and 6th stage larvae of Acheta domesticus were used. Post-flight experiments revealed a low susceptibility of the behavior to micro- and hypergravity while the physiology of the PSI was significantly affected. Immunocytological investigations revealed a stage-dependent sensitivity of thoracic GABAergic motoneurons to 3 g-conditions concerning their soma sizes but not their topographical arrangement. The morphology of neuromuscular junctions was not affected by 3 g-hypergravity. Peptidergic neurons from cerebral sensorimotor centers revealed no significant modifications by microgravity (micro g). The contrary physiological and behavioral results indicate a facilitation of 1 g-readaptation originating from accessory gravity, proprioceptive and visual sense organs. Absence of anatomical modifications point to an effective time window of micro g or 3 g-expo-sure related to the period of neuronal proliferation. The analysis of basic mechanisms of how animals and man adapt to altered gravitational conditions will profit from a continuation of the project "Crickets in Space".     

Environmental impacts on the developing CNS: CD15, NCAM-L1, and GFAP expression in rat neonates exposed to hypergravity.

We have previously reported that the developing rat cerebellum is affected by hypergravity exposure. The effect is observed during a period of both granule and glial cell proliferation and neuronal migration in the cerebellum and coincides with changes in thyroid hormone levels. The present study begins to address the molecular mechanisms involved in the cerebellar response to hypergravity. Specifically, the study focuses on the expression of cerebellar proteins that are known to be directly involved in cell-cell interactions [protein expressing 3-fucosyl-N-acetyl-lactosamine antigen (CD15), neuronal cell adhesion molecule (NCAM-L1)] and those that affect cell-cell interactions indirectly [glial fibrillary acidic protein (GFAP)] in rat neonates exposed to centrifuge-produced hypergravity. Cerebellar mass and protein expression in rat neonates exposed to hypergravity (1.5 G) from gestational day (G) 11 to postnatal day (P) 30 were compared at one of six time points between P6 and P30 against rat neonates developing under normal gravity. Proteins were analyzed by quantitative western blots of cerebellar homogenates prepared from male or female neonates. Cerebellar size was most clearly reduced in male neonates on P6 and in female neonates on P9, with a significant gender difference; differences in cerebellar mass remained significant even when change in total body mass was factored in. Densitometric analysis of western blots revealed both quantitative and temporal changes in the expression of selected cerebellar proteins that coincided with changes in cerebellar mass and were gender-specific. In fact, our data indicated certain significant differences even between male and female control animals. A maximal decrease in expression of CD15 was observed in HG females on P9, coinciding with maximal change in their cerebellar mass. A shift in the time-course of NCAM-L1 expression resulted in a significant increase in NCAM-L1 in HG males on P18, an isolated time at which cerebellar mass does not significantly differ between HG and SC neonates. A maximal decrease in expression of GFAP was observed in HG males on P6, coinciding with maximal change in their cerebellar mass. Altered expression of cerebellar proteins is likely to affect a number of developmental processes and contribute to the structural and functional alterations seen in the CNS developing under altered gravity. Our data suggest that both cerebellar development and its response to gravitational manipulations differ in males and females.