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Hardware and variables: The identical environments of both incubators provided optimal incubation conditions which ensured maximum opportunity to compare flight and ground synchronous chickens. The high percentage of embryos that survived confirmed that the hardware was well designed and functioned properly throughout the duration of the experiment (Figs. 5-8). The eight embryos that were randomly assigned for dissection [of the pre-incubation group (2 days) and flown for 5 days (7 days at landing)] were dead when the shells were open. Autopsy of the embryos revealed that 4 embryos advanced to 6.5, 4, 3.5 and 3 days respectively, suggesting that pre-incubated embryos survived after ascent and exposure to microgravity. It is assumed that microgravity may have affected their development. The remaining embryos showed scattered pigmentation suggestive of lysis after death which probably occurred after the second day pre-incubation period. All space embryos were pre-candled for viability prior to ascent, and only embryos with a high probability of fertility at preincubation were included. Synchronous control eggs were exposed to vibration and gravity forces similar to those produced during the ascent of the space Shuttle (Figs. 6-7), with no adverse effect on viability or hatchability (Fig. 8) of the synchronous chickens.
Thus, unexplained phenomena may act at the molecular level to affect early embryo survival. Protein transport in membrane (not across membranes) could be altered by hormones aided by water transport across the membranes (Haines, 1994). Conductive implants that shunt endogenous electric fields away from the embryo caused developmental defects (Hotary and Robinson, 1992). Changes such as those induced by electric and also ionic changes can affect gene expression leading to change in phenotype (Vandenbroeck et al., 1992). Ultraviolet light on the other hand can, similar to g-radiation, affect avian limb positional signaling, which in turn causes misdirection of the normal limb polarization (Honig, 1982). Moreover, weak, extremely-low-frequency magnetic fields (PMF) have irreversible effects on the early development of (less than 48 hours) chick embryos (Ubeda et al., 1994). The work on PMF indicates that viable fertile eggs exposed during the first 48 hours of post-laying to 100Hz repetition rate, 1.0µT peak-to-peak amplitude, and 500 µs pulse duration)developed numerous abnormalities when surviving to older stages. Cultured cells exposed to electromagnetic fields also responded by altering gene transcription (Phillips, 1993), suggesting that the effect of these environmental variables must be taken into account with experiments using avian embryos, even though variability in avian embryos probably contributes to a source for experimental error in improperly designed studies (Terol and Panchonruiz, 1994). The reason why embryos younger than 48 hours of incubation usually die in space should therefore be investigated by including pre- and non incubated fertile eggs in future flights. Finally, if possible, fertile eggs should be produced in microgravity to develop embryos without the influence of 1.0G. Variables other than those previously examined and controlled for in previous flight experiments with avian embryos must be taken into consideration in the future. All of the eight eggs exposed to the space environment between the 9th and 14 th day of embryogenesis hatched at a time which did not differ significantly from earth-based synchronous controls that were subjected to vibration and acceleration forces (Figs. 6-8). Eggs shells retrieved from the chicks which successfully hatched showed no differences in shell mineral content between treatment. Post-hatch performance, measured in the first and second generation of chicks, was unaffected by exposing the embryos to the space environment (Hester et al., 1993). Since bloodıs packed cell volumes at 9 days of age were significantly lower in space flight chicks as compared to controls, the finding suggests that the effect that microgravity has on the embryonic development may persist for up to two weeks post hatched. This is consistent with the possible effect that microgravity had on the vestibular response measured at 1.0G in hatchlings (Jones et al., 1993).
Temporal bone gross anatomy: Macrocospically, the temporal bones and the membranous labyrinth housed within, appeared normal. However, close examination and morphometric measures of the cells and afferent fibers in the sensory epithelia revealed alterations in the peripheral components (Figs. 16-17 & 24-27) that would affect vestibular function. No electron microscopy observations were done, but high magnifications of light micrographs permitted evaluating alterations of sensory and non-sensory cells. In particular we paid attention to changes in appearance of the cartilage cells that form the otic capsule, because mineralization is under way at the stage examined (Knowlton, 1967; Hogg, 1990). Hypertrophy of the cartilage (Figs. 9-12) which characterizes chondrification (Shinomura and Kimate, 1990), is related to protein matrix glycans (Mann, 1988; Yet et al., 1988), which play an important role in calcification (Shepard, 1992). This is in agreement with our previous hypothesis that a differential distribution of glyproteins and proteoglycans probably contribute to calcification in the inner ear (Fermin et al., 1990; Fermin, 1993). Glycoproteins play specific roles in cartilage homeostasis (Lust et al., 1991), and similar roles could be served in the inner ear otic capsule and calcified membranes (Lim and Rueda, 1990; Santi et al., 1990; Munyer and Schulte, 1991). A lack of significant difference (Figure 13) in the immunoreaction of cartilage cells to anti-keratan, heparan and chrondroitin sulfate between ground and flight temporal bones could reside in inter-individual differences we did not quantitate. It is not uncommon to obtain from the same batch, embryos that differ in their actual gestational age by more than 24 hours (Hamburger and Hamilton, 1951). We staged all embryos examined and determined their age by days to be similar, but could not determine how many hours difference there were between the developmental stage of each embryo (Jones et al. , 1993).
Immunohistochemistry: Surprisingly, the gelatinous, membranes including the statoconial membranes illustrated in Figures 14-15 & 18-19 over the saccular macula, did not react with the glycoprotein fibronectin, the proteoglycan keratan sulfate or chondroitin sulfate (Fermin et al. , 1990; Fermin et al., 1995). The calcifying gelatinous membranes of the maculae of the utricle, saccule and lagena became extremely basophilic with fixation and histological stains used (e.g., hematoxylin & Eosin). It is possible that availability of the glycans above for development and mineralization of otoconia (Fermin et al. , 1995), in the vestibule does not correspond to the availability of these molecules for cartilage mineralization (Hogg, 1990) outside the vestibule. The importance of whether availability of such molecules is concurrent inside and outside the vestibule awaits investigation.
The absence of immuno-reactivity in the otoconia with antibodies previously known to be an integral part of the calcifying gelatinous membranes (Fermin et al. , 1995), could be due to the fact that specimens were kept in the primary fixative for over two months. Antigenic determinants that would react with keratan sulfate fibronectin and chondroitin sulfate in the extracellular otoconial membrane were probably lost to fixative replenishing and rinsing, which was done between specimen collection and processing. We showed before that at the electron microscopy level, antigenic determinants could be removed from their original place and contribute to reaction in non-cellular areas or complete lack of signal in areas of the specimens where one expect to find label (Fermin et al., 1994). Maintenance of specimens for over 2 months in the primary fixative was necessary because the experiment was double-blinded. Under these experimental conditions, financial resources available for the project did not permit examining all 16 specimens at once. In addition, cutting pieces from whole temporal bone for EM, while leaving the remaining tissues for histology, would have disrupted the normal organization of inner ear tissues. Thus, we opted for embedding all temporal bones in paraffin which allowed histological and immuno-histological processing (Figs. 9-12, 14-19 & 24-27).
The cartilaginous otic capsule has tightly bound cells (chondrioblasts, fibroblasts, etc.) in the hyaline matrix. In such a matrix, molecules of interest or antigenetic determinants for the above antibodies were not lost. Keratan sulfate, chondroitin sulfate and fibronectin were present in the cartilaginous otic matrix after the extended fixation protocol mentioned above, and were as normally found, in the vestibular apparatus (Igarashi et al., 1993). The hyaline cartilage of the inner ear serves normally as a built in positive control for polylactosaminoglycans (Fermin et al. , 1990; Fermin et al. , 1995). Thus, the cartilaginous otic capsule was examined for keratan sulfate (Jalkanen, 1987), fibronectin (Lust et al. , 1991) and chondritin sulfate (Bonucci and Silvestrini, 1992; Yamagata et al., 1993) immunostains. These molecules are very important in cartilage calcification (Kogaya et al., 1987; Shinomura and Kimate, 1990). Cartilage chondrification and bone formation occur around E14, and changes in the chondrification and mineralization were reported earlier (Knowlton, 1967).
From the sixth to the eighth day of incubation the entire neurocranium is laid down as a continuous mass of cartilage (Hogg, 1990). No significant difference of the otic capsule cartilage cells between flight and ground controls (Figs. 11-12) was found, even though we anticipated the opposite. Particularly, because formation of the correct skeletal pattern depends on the cell condensation process that is evident in hypertrophy of the cartilage. Usually, direct cell substrate or cell-cell interactions plus signals in the cartilaginous matrix may be involved in the regulation of the condensation process (Shinomura and Kimate, 1990). This is because during condensation the mesenchymal cells undergo a shape change probably due to the physical stresses induced by transient signals. Re-absorption of the dense mesenchyme in some areas of the otic capsule gives origin to the vestibule proper (Knowlton, 1967).
Calcification of the cartilage begins around stage 38 and continues through the time we collected the embryos (Jaskoll and Maderson, 1978; Hogg, 1990). Since calcium has been shown to be actively mobilized during space flights (Ross, 1987; Vico et al., 1987; Pozharskaya and Noskov, 1990; Rehak et al., 1991), modifications that microgravity induce to chondrifying cartilage could affect the morphometry of cells involved in chondrification and calcification of the otic capsule that encapsulate the inner earıs delicate membranes. If such is the case, alterations of developmental molecular mechanisms leading to chondrification could occur, but not show up histologically unless biochemical analysis of specific macro-molecules in question is conducted. Instead we applied immunohistochemical assays to test for macro-molecules present in the cartilaginous matrix. Keratan sulfate and chondroitin sulfate are abundant in normal cartilage. The structural integrity of collagen network and that of proteoglycans are important in maintaining the stiffness and resiliency of cartilage. Ideally, an hour per hour match between the embryos compared should exist, but such analysis was not possible under the experimental design utilized. Immunoreactivity of cartilage cell for each antibody (n=300) is shown in Figure 13. Keratan sulfate and chondroitin sulfate, known components of hyaline cartilage seemed more abundant than fibronectin. However, the distribution pattern (patches) of chondrioblasts undergoing modification which precede ossification in other systems, was different among the embryos. Yet, as mentioned before this non-significant difference observed could be due to interindividual variability.
Table 3 shows the relative abundance of patchiness (Figs. 9-10) observed on the cartilaginous otic capsule with trichrome and immunohistological stains. The average measurements of patchy vs. non-patchy areas chosen at random yielded approximately 60:90 ratio of cells per screen full at x160 magnification at the camera chip and over 2,000 magnification at the video screen. Cartilage cells rotated inside the cartilaginous matrix and increased their sizes during hypertrophy, but their shapes were not altered (Figs. 24-25). The parameters measured above failed to show significant alterations of inner ear structures of space flown chicks. However, one must remember that surviving embryos used for our study were pre-incubated at 1.0G up to the time when differentiation of inner ear vestibular end organ occurs. Thus, cells in those organs could already have been exposed to the signals that at 1.0G set the progression for normal uninterrupted development in modified gravity. Lastly, immuno-histochemical stain for keratan sulfate, chondroitin sulfate and for fibronectin indicated that these macromolecules of the cartilage provide good indicators of cartilage modifications including chondrification and other processes that precede ossification. Modifications of enzymatic and metabolic pathways may occur transiently, but may escape our present methods of analysis. It will be interesting to see in future experiments whether cellular and molecular techniques show significant differences in the inner ear tissue of ground and flight groups.
The gross morphological changes observed in the epithelia were small and mostly non-signficant at the level of confidence tested (Figs. 20-21). This was surprising because we expected to see great variability in the gravity organs of the inner ear between flight and ground controls. It seems that the changes induced by exposure of the living organism to microgravity are subtle and probably reversible, even more so in developing systems which have greater plasticity than mature systems (Crossland, 1981; Cotman, 1990; Fazeli, 1992). The height of the epithelia (Fig. 21), the change in location of the support cellsı nuclei and their density, and vasculorization of the cytoplasm are probably reversible alterations that revert to normal after a certain period. Similarly, the observed difference in the number of afferent fibers stained with anti-neurofilament antibody may represent a temporary change (Figs. 23-27) that was previously observed in the inner ear (Ross, 1994), the brain cortex synaptic pattern (Dyachkova, 1991), and neurotransmitter receptors (Miller et al., 1985) of rodents exposed to microgravity. Nonetheless, the change in peripheral afferents probably induces alterations in the synaptic connectivity at the brain stem level (Precht et al., 1966; Darlington et al., 1991; Imate and Sekitani, 1993; Claussen, 1994), which in turn may affect other parts of the vestibular pathways (Lorente De Nó, 1933; Brodal et al., 1962). It is clear from our observations and those of other investigators that more work is needed before the contribution of each individual vestibular component (Fig. 4) is fully understood in relation to total vestibular function.
The changes in the branching of the afferents could represent one of the most significant changes of the vestibular system in response to change in gravity. Modifications of the afferent system may contribute to motion sickness (Lackner et al., 1991; Golding, 1992; Hlavacka et al., 1992; Takahashi et al., 1992), particularly as it relates to the control of posture (Lacour and Borel, 1993). Visually induced motion sickness is well characterized and known to be so important that even patients with bilateral vestibular defects are made sick (Cheung et al., 1991) by strong visual stimuli. The embryos were not exposed to visual stimuli, and we assume that changes observed were vestibular. Surviving embryos that returned on the STS-29 and were allowed to hatch showed altered responses (Jones et al. , 1993) weeks after landing and assumed readaptation to 1.0G. Latency and amplitude data alone may not show significant space flight effects (Table 4). However, the mixed results for thresholds and the abnormal responses in 3 of 8 flight animals suggested that at least some of the animals were affected by space flight. It is possible also that all animals were affected originally by the flight and that only three of these animals failed to recover normal thresholds over the 28 days of adaptation following the mission. No morphological data are available for the chicks that hatched and were measured for vestibular function. However morphological findings for flight embryos of the same study suggest that changes in nerve branching could influence funtional thresholds. Although it was not possible to test the embryos behaviorly or functionally before re-exposing them to 1.0G, the abnormal afferent branching observed in embryos fixed a few hours after landing (Fig. 23) may require time for repair at 1.0G for the embryos that were allowed to hatch. If repair (or a reversion to 1.0G pattern) lasted for more than two weeks, the altered vestibular response recorded post-hatch (Fig. 28) could have resulted from rewiring and/or modifications of synaptic contacts. Rodents vestibular organs undergo similar changes (Ross, 1994), and the propensity of humans to develop motion sickess is greater in microgravity than at 1.0G. Re-wiring of afferent fibers and modification of synpatic contacts in the vestibular inner ear organs may occur in humans exposed to microgravity. The possibility that the avian vestibular system responds to microgravity in similar manner as mammals do, warrant further analysis of the avian vestibular system after exposure to microgravity. Avian embryos do not require dependancy of a pregnant mother, with obvious implications for waste removal, biomass utilization and containment.
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