Chapter 10

General discussion

10.1 Hypergravity and bodyweight

Before discussing the results of the behavioural and structural data, the effect of hyper-gravity on the body weight will be evaluated. As reported in the previous chapters (chapters 4, 6 and 9) long term exposure to hypergravity resulted in a decreased body weight in hamsters exposed to hyper-gravity during embryonal development as well as for hamsters subjected to hyper-gravity after birth. This decrease is solely caused by the increased gravity forces, not by the rotational accelerations experienced by the hamsters during centrifugation. Hamsters placed in the center of the centrifuge (the rotation group, chapter 9) grew as fast as control hamsters. Furthermore, this weight decrease was not dependent upon whether an animal was conceived in hyper- or normal gravity. The body weight increase was the same for both groups (HG animals of chapter 6 and 9) during 4 months of hypergravity and they weighted the same after this period. The differences in body weight between groups and its effect on certain perceptive motor skills, such as balancing on tubes, will be discussed in the following paragraph.

10.2 Functional adaptation to hypergravity

To detect differences in the function of the peripheral vestibular system between hamsters exposed to hypergravity and control hamsters living in normal gravity, the hamsters were subjected to a variety of tests which assessed vestibular evoked or controlled behaviour. The first goal was to develop new tasks or to modify existing ones to test perceptive motor skills. The tests used in the pilot experiment (chapter 4), i.e. balancing on tubes, swimming behaviour and open field activity were useful for detecting differences in behaviour between HG hamsters and controls. For some tasks, e.g. swimming behaviour, differences between groups remained for months. Together with the air-righting responses, treadmill activity and susceptibility for rotations, the effects of hyper-gravity on vestibular controlled behaviour could be examined.

Normal locomotion was not affected by either hypergravity or rotation (table I). The hamsters subjected to hyper-gravity or rotation walked normally, just as the control hamsters, while human subjects showed postural and locomotor disturbances after an increase or a decrease in gravity especially when making head movements (Clement et al. 1985; Young et al. 1986; Bles et al. 1991; Bles and de Graaf, 1993; Paloski et al. 1993). A decreased rearing activity in the open field was found in the HG developed hamsters when compared with the other groups, indicating that these hamsters were less eager to make vertical movements in which the heads were turned. A similar decrease in rearing activity was also found in labyrinthectomized rodents (Ossenkopp et al. 1990; Ossenkopp et al. 1992). It is difficult to say whether this decrease in rearing activity can be compared with the decrease in head movements in human subjects that was observed after an increase or a decrease of gravity (Young et al. 1986; Ble-s et al. 1991; Bles and de Graaf, 1993; Paloski et al. 1993). If these behavioural patterns are the same indeed, decreases in rearing activity in hamsters subjected to hypergravity after birth were to be expected, which was not found (Chapter 6). Because the rotation hamsters acted the same as controls it can be concluded that the otolith system is responsible for the decrease in vertical activity. Therefore, it is suggested that the decreased rearing activity of the HG developed hamsters was the result of structural alterations in their otolith organs.

When the animals were subjected to locomotor tasks where achievement dependent largely on vestibular information (as in the tube tasks) the HG hamsters performed worse than control hamsters. During hypergravity the otolith organs are continuously and vigorously stimulated which reduces their response not only during hypergravity but also immediately after return to normal gravity (Welch et al. 1996). This decreased responsivity affects, among others, equilibrium maintenance on the tubes. The results from the hamsters subjec-ted to rotation (in a normal gravity environment) showed that also these hamsters had a deteriorated performance on the tube tasks resembling the performance of HG hamsters. The somatosensory information was not altered in this group indicating that the balance disturbances were not caused by the effect of increased gravity forces on somatosensory information. The body weight affected performance in the tube tasks when the hamsters grew bigger by altering their body dimension resulting in a change in the center of gravity. The control hamsters and the hamsters subjected to rotation, which grew faster than HG hamsters, had more difficulties with balancing -than HG hamsters at the end of the experiments. In the first months of the experiment, however, no differences in body weight were found between HG hamsters and controls. This shows that balance disturbances during the first months of the experiments were not caused by differences in body weight.

The mobile tube task was introduced because it was expected that the performance on this tube was even more dependent on vestibular information than on the fixed tube. The results showed that balancing on the mobile tube was more difficult than on the fixed tube. However, not only the HG groups had more difficulties with this task but the control and rotation groups as well, indicating that no extra information was acquired by subjecting the animals to this task.

Differences in performance of the tube tasks disappeared when the hamsters were living in normal gravity conditions. Quick normalization of equilibrium maintenance on rails after placement in 1 G was also observed in rats exposed to 2 G for 60 days (Clark, 1973, 1974). Moreover, hamsters conceived in hyper-gravity and subjected to normal gravity after weaning (HC group, chapter 9) did not have difficulties with keeping balance. These findings indicate that these disturbances are temporary and only occur immediately after ending the accelerative forces, i.e. during centrifuge stops. It is concluded that the observed disturbed equilibrium maintenance on the tubes is caused by an alteration in vestibular input.- But it remains unknown whether this disturbance was caused by the changes in otolith information alone or by a change in both canal and otolith information.

As was concluded in chapter 4 and 6, training also enhanced performance on the tube tasks. During training the animals seem to become more dependent on visual and somato-sensory information, for example, tactile information coming from their paws. Astronauts in space and after return to earth, also became more dependent on somatosensory information (sway-references to foot support) for recovery of postural control in normal gravity (Young et al. 1986; Lipshits et al. 1994; Black et al. 1995). Furthermore, some human subjects became more dependent on visual information after a hypergravity or a micro-gravity period (Clement et al. 1985; Bles and de Graaf, 1993). The results of the hamster experiments support the suggestion made by other investigators (Ockels et al. 1990; Bles and de Graaf 1993; Lipshits et al. 1994; Black et al. 1995) that subjects become more dependent on somatosensory information rather than vestibular information after microgravity or hypergravity. Furthermore, the data confirm the suggestion made by Money (1991) that in the CNS, vestibular information is either ignored or suppressed and substituted by information from the other sensors during prolonged altered gravity states regardless of whet-her this is an increase or a decrease in gravity.

Training on the tubes did not enhance performance in the adult hamsters subjected to hypergravity. Even after 2 months, 50% of these HG hamsters were unable to walk on the tube (chapter 7). This outcome suggests that age is a factor for the adaptation mechanism to accelerative forces, confirming the conclusion of earlier studies concerning decreasing adaptive capacities of the vestibular system in aging humans (Oosterveld, 1983; Paige-, 1991).

The most pronounced effect of hypergravity on swimming behaviour was found in the hamsters which were conceived, born and raised in 2.5 G (HG hamsters of chapter 9). The swimming ability was severely disturbed in these hamsters. Normal swimming requires multisensory integration and a rapid processing of information (Petro-sini, 1983). One could attribute this inability of HG developed hamsters to swim normally to their lack of body fat, which normally facilitates floating ability. If this was true, than the hamsters exposed to hypergravity after birth (chapter 6), which weigh-ted the same as hamsters conceived in hypergravity, should have shown the same swimming deficit. But, these hamsters swam almost as fast as control hamsters and no differences were found in their swimming movements. With respect to spatial orientation, however, the results from the water-maze suggest that these HG hamsters also had problems with orientation during swimming in normal gravity. -The severe swimming disturbances of the HG conceived hamsters might be attributed to structural adaptations in the peripheral system in this group.

Problems with spatial orientation or alterations in spatial orientation were also detected in the number of correct air-rightings and the number of turnings in the rotation task. Again, the HG developed hamsters living in hypergravity showed fewer correct air-righting responses than hamsters subjected to hyper-gravity after birth. The number of turnings decreased equally for both prenatal and postnatal exposure to hyper-gravity. Rotation alone (hamsters in the center of the centrifuge, chapter 9) had no effect on either the number of correct air-righting-s responses or the number of turnings, thus showing that the increased gravity forces are responsible for these effects. The disturbed swimming ability and air-righting reflex and the decreased number of turnings indicate an alteration in vestibular sensation. As was reported earlier in chapter 9, rats with lesions in the vestibular labyrinth also showed disturbances in these behaviours (Ossen-kopp et al. 1992).

When the HG hamsters were living in normal gravity following the hyper-gravity period, differences in swimming, air-righting and turning behaviour remained, indicating a persistent behavioural adaptation to hypergravity during normal gravity. Again, the hamsters developed in hyper-gravity showed more persistent disturbances in behaviour than hamsters placed in the centrifuge after birth. It is suggested that the structural alterations in the peripheral vestibular system (in particular the otolith organs) cause these alterations in behaviour. As will be reported in the next paragraph, alterations were found in the otoconial layer as well as in the sensory epithelium of the otolith organs.

It is concluded that a functional adaptation of the vestibular system is one of the causes for the disturbances in vestibular evoked or controlled behaviour. The severity of disturbances in spatial orientation are dependent on the environment in which embryonal development takes place and on the age of the animals when subjected to altered gravity. Furthermore, hypergravity and rotation have a different effect on vestibular evoked behaviour.

Table 1: Summary of all the behavioural tests to evaluate the performance of the perceptive motor skills. Brackets = (conceived and born in ..G tested in ..G), # = not tested in this group, 0 = not different with controls, x = worse or decrease performance when compared to controls, xx = task for 50% of the group too difficult.

10.3 Hypergravity and morphological alterations in the peripheral vestibular system

Longterm exposure to hypergravity after birth does not result in structural alterations in the otoconial layer of the utricle and saccule (chapter 5, chapter 7). However, exposure to HG during the formation of the otoconia changes the relative size of areas occupied by large, medium-sized and small otoconia on the utricular macula. The growth of some areas- see-m-s to be delayed (chapter 8). Differences in otoconial distribution still exists after 8 months of normal gravity suggesting that the adaptation is irreversible. The Ca2+ content, size or shape of the otoconia was the same in all groups, indicating that otoconia formation is the same during hyper- and normal gravity and that gravity alterations do not change the otoconia morphology.

Structural adaptation may also take place at the sensory epithelium of the otolith organs. As a pilot experiment, the otolith organs of HG and control hamsters (young hamsters = 21 days and adult hamsters = 8 months) were prepared to study the vestibular sensory epithelium, especially the cytoskeleton of the cells, with confocal microscopy and scanning electron microscopy. Because the findings of this experiment should be regarded as preliminary ones, only a brief presentation is given. The cytoskeleton of the cell is responsible for cell morphology and internal organization (Taku-mida et al. 1995). Therefore, a study of the cytoskeleton can enhance our knowledge about the functional properties of the cells of the vestibular system and how hyper-gravity affects these properties. The cytoskeleton was examined with immunochemistry and confocal microscopy. The microtubule and actin content of the hair cells, hair bundles and supporting cells in the vestibular epithelium were assessed in young and adult HG and control hamsters.

It was found that the density of hair cells and supporting cells of the maculae was the same in the striola and peripheral region for the young and adult control hamster. The number of supporting cells was larger for the young hamster than for the adult one. Furthermore, the apical Microtubule (MT) ring was larger in the striola region than in the peripheral region in the young hamster, while the opposite was found in the adult. Large sized supporting cells were found in the peripheral region and smaller ones around the striola. This difference was even bigger in the young hamster. The young hamsters also differed from the adults in the size of the actin layer surrounding the supporting cells which was much thicker in the adult hamsters. No differences were found in the size of the hair bundle base size and in the distance from the apical MT ring to the microtubule network. These results indicate that during maturation the skeletal actin in the supporting cells thickens and the number and size of the supporting cells decrease.

The density of hair cells and supporting cells in the striola region was the same for the young and adult HG hamster. However, in the adult peripheral region there were less hair cells than in the young one. The size of the apical MT rings and the size of the supporting cells was larger in the striola region than in the peripheral region. The size of the actin layer was thicker in the adult hamsters and no differences were found hair bundle base size. It is concluded that the skeletal actin thickens in the adult HG hamster just as in the adult CON hamster.

When comparing the HG hamsters with their age-matched controls, it was found that the adult control hamsters had more hair cells and less supporting cells in the striola region than the HG hamsters. No differences in hair cell/supporting cells ratio were found between young HG and control hamsters. The apical MT ring was larger in the striola region than in the peripheral region in the young and adult HG hamster, while the opposite was found in the normal hamsters. The size of the supporting cells was larger in the peripheral region than in striola region except in the young HG hamster in which the size was the same in both regions. Furthermore, the size of the actin layer was significantly smaller in the HG hamsters than in the age-matched controls. The size of the base of the hair bundle and the distance from the apical MT ring to the microtubule network was the same in all groups. These results indicate an alteration in the maturation of the vestibular end-organ when hamsters are exposed to hypergravity during development. This results in a thinner actin layer and less sensory hair cells with increasing age.

10.4 Recommendations for future research

In the experiments of this thesis, the eye movements of the animals and the effect of hyper-gravi-ty on these movements were not studied. The function of peripheral vestibular system can- be tested most directly by means of the vestibulo-oculomotor reflex (V.O.R.). The movement of the eyes which are assessed by this reflex, represents the function of the vestibular system. With the V.O.R. reflex the relationship between structure and function of the vestibular system can be studied extensively during hyper-gravity, normal gravity and microgravity. Instead of using hamsters, rats are recommended for this kind of study. The Magnetic Field Coil system, which examines the eye movements, is described as being very accurate in rats (Kasper et al. 1987; Dieringer and Meier, 1993). The movements can be recorded with search coils (removable) implanted on the eyeballs of the rats. The V.O.R. can then be recorded during hypergravity, normal gravity and during rotations in normal gravity. T-he rats would be subjected to microgravity, by means of parabolic flights. During these flights the V.O.R. reflex would be studied during weightlessness, normal gravity and hypergravity and compared with the results obtained from experiments with humans during parabolic flight campaigns.

The results of the study of the vestibular sensory epithelia, described in the last paragraph, must be regarded as preliminary. Further anatomical investigations of the otolith organs are necessary to assess and confirm the structural tissue changes in the sensory epithelium as described in the pilot experiment discussed in paragraph 10.3. Therefore, examination of the cells of the epithelia by means of scanning electron microscopy (shape, number and distribution of cilia, haircells and supporting cells), transmission electron microscopy (internal organization of type I and II hair-cells and supporting cells) and confocal laser scanning microscopy (intracellular changes, actine and microtubuline filaments) is suggested. These methods can also be used to assess possible structural alterations in the sensory epithelium of the semicircular canals.