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The influence of gravity on the process of development of animal systems.

The development of animal systems is described in terms of a series of overlapping phases: pattern specification; differentiation; growth; and aging. The extent to which altered (micro) gravity (g) affects those phases is briefly reviewed for several animal systems. As a model, amphibian egg/early embryo is described. Recent data derived from clinostat protocols indicates that microgravity simulation alters early pattern specification (dorsal/ventral polarity) but does not adversely influence subsequent morphogenesis. Possible explanations for the absence of catastrophic microgravity effects on amphibian embryogenesis are discussed.

Animals↗

Amphibian egg cytoplasm response to altered g-forces and gravity orientation.

Elucidation of dorsal/ventral polarity and primary embryonic axis development in amphibian embryos requires an understanding of cytoplasmic rearrangements in fertile eggs at the biophysical, physiological, and biochemical levels. Evidence is presented that amphibian egg cytoplasmic components are compartmentalized. The effects of altered orientation to the gravitational vector (i.e., egg inversion) and alterations in gravity force ranging from hypergravity (centrifugation) to simulated microgravity (i.e., horizontal clinostat rotation) on cytoplasmic compartment rearrangements are reviewed. The behavior of yolk compartments as well as a newly defined (with monoclonal antibody) non-yolk cytoplasmic compartment, in inverted eggs and in eggs rotated on horizontal clinostats at their buoyant density, is discussed.

Animals↗

Interaction of growth-determining systems with gravity.

The experiments have been carried out with lettuce shoots on board the Salyut-7 orbital station the Kosmos-1667 biological satellite and under ground conditions at 180 degrees plant inversion. By means of the centrifuge Biogravistat-1M the threshold value of gravitational sensitivity of lettuce shoots has been determined on board the Salyut-7 station. It was found to be equal to 2.9 x 10(-3)g for hypocotyls and 1.5 x 10(-4)g for roots. The following results have been received in the experiment performed on board the Kosmos-1667 satellite: a) under microgravity the proliferation of the meristem cells and the growth of roots did not differ from the control; b) the growth of hypocotyls in length was significantly enhanced in microgravity; c) under microgravity transverse growth of hypocotyls (increase in cross sectional area) was significantly increased due to enhancement of cortical parenchyma cell growth. At 180 degrees inversion in Earth's gravity root extension growth and rate of cell division in the root apical meristem were decreased. The determination of DNA-fuchsin value in the nuclei of the cell root apexes showed that inversion affected processes of the cell cycle preceding cytokinesis.

Cell Division↗

Formation and growth of callus tissue of Arabidopsis under changed gravity.

The changed gravity conditions do not prevent the process of cell dedifferentiation and formation of callus. Yet, callus grown on the clinostat and in space differs from the control one: its consistence is denser, occurence of meristematic centres is less frequent and it shows a reduced proliferative activity of cells. Average size of cell and nuclei area in the test variants is lower than in the control.

Arabidopsis↗

Evolution of a phase separated gravity independent bioreactor.

The evolution of a phase-separated gravity-independent bioreactor is described. The initial prototype, a zero head-space manifold silicone membrane based reactor, maintained large diffusional resistances. Obtaining oxygen transfer rates needed to support carbon-recycling aerobic microbes is impossible if large resistances are maintained. Next generation designs (Mark I and II) mimic heat exchanger design to promote turbulence at the tubing-liquid interface, thereby reducing liquid and gas side diffusional resistances. While oxygen transfer rates increased by a factor of ten, liquid channeling prevented further increases. To overcome these problems, a Mark III reactor was developed which maintains inverted phases, i.e., media flows inside the silicone tubing, oxygen gas is applied external to the tubing. This enhances design through changes in gas side driving force concentration and liquid side turbulence levels. Combining an applied external pressure of four atmospheres with increased Reynolds numbers resulted in oxygen transfer intensities of 232 mmol O2/l/h (1000 times greater than first prototype and comparable to a conventional fermenter). A 1.0 liter Mark III reactor can potentially deliver oxygen supplies necessary to support cell cultures needed to recycle a 10 astronaut carbon load continuously.

Atmospheric Pressure↗

Gravity effects on membrane processes.

Application of the Gouy-Chapman-Debye-Hückel (GCDH) theory to a model membrane in contact with electrolytes of various concentrations and composition predict density variations within an interfacial layer. Assuming that on cellular dimensions hydrodynamics can be applied (the objections are briefly discussed) two types of gravity effects can be defined, 1. convection along the surface of vertically oriented membranes and 2. surface potential variations by layer deformations at horizontally oriented membranes. Both effects should affect transport across the layer to the membrane surface and across the membrane. According to the theoretical predictions first experiments with gramicidin channels incorporated into artificial phosphatidylserine bilayer membranes show a significant difference in single channel currents in vertical and horizontal membranes. The complexity of biological membrane functions requires investigation of isolated membrane surface reactions and transport systems to study the gravisensitivity for each process separately.

Cell Membrane↗

Xenopus laevis embryos can establish their spatial bilateral symmetrical body pattern without gravity.

One assumes that gravity cooperates with the sperm in the establishment of bilateral symmetry in the embryo, particularly in species with yolky eggs. However, only experiments under genuine microgravity can prove this. May 2nd 1988 on the TEXUS-17 Sounding Rocket, eggs of Xenopus laevis became the first vertebrate eggs ever successfully fertilized in Space. Fertilization was done in fully automated hardware; the experiment was successfully repeated and extended in 1989. Here we report a "Space First" from the IML-1 Space Shuttle mission (January 1992): In similar hardware and under microgravity, artificially fertilized Xenopus eggs started embryonic development. Histological fixation was pre-programmed at the time gastrulation would occur on Earth and indeed, gastrulae were fixed. Thus after fertilization in near weightlessness Xenopus embryos do develop bilaterally symmetrically, very probably cued by the sperm alone.

Animals↗

A direct approach to the study of the effect of gravity on axis formation in birds.

A system has been developed to enable the normal development of aborted very early uterine avian embryos, outside the female's uterus. The shell-less aborted egg was put into a foster shell of a sister egg, previously laid by the same female. The empty space between the shell and aborted egg was filled with artificial uterine fluid. The reconstructed eggs were incubated at 42 degrees C for 30 hours in a vertical position. The atmosphere contained a high concentration of CO2 (8-10%). At the termination of the 30 h the eggs were transferred to incubation at 37 degrees C in normal atmospheric conditions. Normal development has been recorded for a certain percentage of eggs incubated up to 12 days. In other cases abnormalities, arrested development or development of extraembryonic membranes only, without a sign of an embryonic axis, have been observed. The three important conclusions from the above experiments were: 1. It is possible to develop a closed, self-contained system, disconnected from the female's body, that would support the development of early uterine embryos. 2. The incidence of embryo-less extraembryonic membranes in such a system, is correlated with the degree of detachment of the "yolk" from the outer envelopes. 3. Such a system can be further developed into an experiment suited for microgravity conditions which will be an alternative to an experiment with live birds. The experiment will be aimed at testing the importance of gravity in changing the radially symmetrical avian blastoderm into a bilaterally symmetrical blastoderm.

Animals↗

Gravity effects on connective tissue biosynthesis by cultured mesenchymal cells.

Quantitative and qualitative aspects of collagen synthesis under microgravity, normal gravity and hypergravity conditions were investigated during the spacelab D-2 mission by incubating human fibroblast cultures with [3H]-proline for 0, 4, 7, 10 and 20 hours. Quantitative analysis revealed an increase of collagen synthesis under microgravity conditions, being 40% higher than 1g controls. Hypergravity samples at 1.44g, 6.6g and 10g showed a decrease in collagen synthesis with increasing g, being down to about 15% at 10g. The relative proportion of collagen from total protein synthesized, the secretion of collagen by the cells, proline hydroxylation of individual collagen alpha-chains and the relative proportions of collagens I, III and V synthesized were not affected at any of the applied conditions.

Cells, Cultured↗

Simulated micro-gravity and cortical inhibition: a study of the hemodynamic-brain interaction.

The present study was carried out to determine the inhibitory cortical processes induced by changes in hemodynamics. Previous experiments in humans conducted in our laboratory have shown that there is a close relationship between posture and delta and theta EEG activity. The most pronounced effects were obtained during the 6 degrees head-down tilt (HDT) position. In space medicine the HDT procedure is very frequently employed to simulate micro-gravity and to determine the neurohormonal counter-regulations evoked by the expansion of central volume. Twenty male subjects spent 23 h in bed in 6 degrees HDT and 23 h in 6 degrees HUT (head-up tilt) positions during which EEG (frontal, central, parietal, occipital), startle responses, and reaction-times were measured every 2 h (from 10:00 h till 20:00 h). The effects of cardiovascular deconditioning (CD) regularly occurring after HDT were assessed by examining orthostatic tolerance and the physical work capacity (bicycle ergometry). As expected, 23 h HDT led to more pronounced CD than HUT. Spectral power analyses of EEG revealed increases in delta and theta frequency hands similar to those found during HDT in previous EEG studies. In addition, subjects responded more slowly (S1-S2 reaction-time task) during HDT as compared with HUT bedrest. The influence of HDT on startle response, however, was not in keeping with the initial hypothesis (i.e. dampening of reflex activity). The EEG data and the sensorimotor performance indicated that the body fluid shift towards the thoracic cavity induced by HDT resulted in signs of cortical inhibition. In addition to neural mechanisms, other processes must be postulated which are closely related to the counter-regulation evoked by the varying body positions.

Adult↗

Separation of intact rat hepatocytes and rat liver nuclei into ploidy classes by velocity sedimentation at unit gravity.

A system is described which permits the separation of isolated hepatocytes and isolated rat liver nuclei belonging to different ploidy classes by velocity sedimentation at unit gravity. The problem of obtaining single cells suspensions is discussed and preparations were obtained that contained 96% single hepatocytes. By improving the sedimentation method, it took 2.5 h to separate rat liver nuclei on sucrose gradients into diploid and tetraploid ploidy classes. Recoveries were generally over 95%. The diploid band was 99% pure. DNA and protein content of the ploidy classes were measured. After partial hepatectomy and [3H]thymidine injection it was found that the label moved largely into the tetraploid compartment. Isolated hepatocytes were fractionated in 1 h on Ficoll gradients. Erythrocytes were separated from small nucleated cells and the population of hepatocytes was clearly separated from these two cell populations. Diploid hepatocytes were 80% and tetraploid hepatocytes were 99% pure. Viability was about 80% after fractionation. The gene dosage of NADPH cytochrome c reductase, succinate dehydrogenase and lactate dehydrogenase was estimated in diploid and tetraploid hepatocytes. Gene dosage was equal in diploid and tetraploid hepatocytes for succinate dehydrogenase and NADPH cytochrome c reductase. It is suggested, after correcting for non-viable tetraploid hepatocytes, that the gene dosage of lactate dehydrogenase was significantly lower in diploid than in tetraploid hepatocytes.

Animals↗

The central afferent and efferent organization of the gravity receptor system of the statocyst of Octopus vulgaris.

In Octopus vulgaris, the projections of the afferent fibers from, and the locations of cell bodies of the efferent fibers of, the nerve innervating and macula (statocyst gravity receptor epithelium) were studied within the CNS using iontophoretic whole nerve injection of either cobalt chloride or Lucifer Yellow CH.13 Afferent fibers from the macula nerve project to subesophageal, periesophageal and supraesophageal areas of the brain. Large numbers of such fibers were found in the subeosophageal lateral pedal and posterior lateral pedal lobes, palliovisceral lobe, and the magnocellular commissure. Afferent fibers were also found in the periesophageal ventral and dorsal magnocellular lobes. Supraesophageal macular nerve afferent projections were seen to the peduncle lobe and the ipsilateral median basal lobe. There is evidence for two different macular nerve afferent projections to the contralateral median basal lobe, via and suprapedal commissure and the macula-to-contralateral-median-basal-lobe tract. Evidence is presented for the location of at least some of the macula's efferent fiber's cell bodies in the lateral and posterior lateral pedal lobes, and magnocellular lobes. The differences between the results obtained here with the two different staining methods are discussed. The results imply that processing of static information occurs in many areas of the Octopus CNS, and is much more complex than previously thought. Some of the possible physiological consequences are considered.

Afferent Pathways↗

Dosimetry of rotational photon fields with gravity-oriented eye blocks.

Evolving radiotherapeutic technique is aimed at routine use of an eye protection accessory with uniformity of dose to facial tumors. Rotational 4 MV photon fields with gravity-oriented eye blocks are described in applications to patients and a phantom. A machine-oriented compensator with a slit and beveled edges at eye levels has been applied to increase uniformity of dose over the entire target, including partly blocked regions. Treatment planning parameters are derived from simple geometric relationships. Dose distributions and average tissue-air ratios (TAR) are calculated with a conventional radiotherapeutic treatment planning system, and measured during initial treatments via thermoluminescent dosemeters (TLD) in facial cavities. Machine monitor units of initial treatments (MU1) are calculated from prescribed dose D, TAR, percentage isodose lines, dose rates in air, and compensator transmission; for subsequent treatments, MU1 are modified using averages of unblocked target doses D measured during initial treatment: MU2 = (D/D)MU1. Doses measured on patients' eyelids have been 10 to 15% of prescribed dose. Three dimensional dose distributions were determined using TLD in an anthropomorphic phantom. With beam compensation, eye doses were 15% of unblocked target doses, doses to partly blocked target were 86 to 129% of unblocked target doses, and doses to unblocked target showed 5% mean standard deviations. In vivo and in phantom data were consistent with computer-calculated distributions. For uniformity of target dose, eye-sparing, and simplicity of irradiating tumors surrounding the eyes, the technique investigated compares favorably with conventional techniques.

Elementary Particles↗

Intraarticular pressures in a gravity-fed arthroscopy fluid delivery system.

Seven consecutive patients undergoing arthroscopic surgery in a gravity-fed fluid delivery system were studied. The average minimum adequate intraarticular pressure (MAIP) was 55 mm Hg. The height of a saline bag above the knee necessary to achieve this average MAIP was 75 cm. There was a positive correlation between diastolic blood pressure and the minimum adequate intraarticular pressure in this study. There was no positive correlation between systolic blood pressure and the MAIP.

Adolescent↗

Effect of gravity on lymphocyte proliferation.

Reports on postflight examination of lymphocytes from crew members of soviet and american spaceships show a depression of reactivity towards mitogens in vitro. The purpose of this communication is to present experimental evidence that gravity can interfere with lymphocyte activation. Lymphocytes were incubated in the presence of concanavalin A in a centrifuge at 2 and 4 g for four days. This environment causes a significant acceleration of the response to the mitogen. In addition, remarkable differences in the ultrastructure of cells grown at 1 g and 4 g are observed by electron microscopy. This investigation is related to the experiment "Effect of weightlessness on lymphocyte proliferation" experiment which will be performed during the first Spacelab mission.

Animals↗

Physiological reactions during acute adaptation to reduced gravity.

Experiments were carried out on healthy volunteers exposed to a 7-day water immersion. The subjects were kept dry by use of a water proof, highly elastic cloth. During the experiment different cardiovascular, fluid-electrolyte and biochemical parameters were recorded. During the 7-day immersion, physiological parameters changed on a phasic basis. An acute period of adaptation began within the first minutes of immersion and ended by the third day. Later some of the parameters showed relative normalization. The experimental results suggest that the hypophyseal-adrenal system and renal function are closely coupled regulatory mechanisms for adaptive reactions of the cardiovascular system and for fluid-electrolyte metabolism during acute adaptation of the human body to reduced gravity.

Adaptation, Physiological↗

Reliable acetabular cup orientation with a new gravity-assisted guidance system.

Acetabular cup orientation is a key factor determining hip stability, and standard mechanical guides have shown little help in improving alignment. An in vitro study was carried out to compare the accuracy and precision of a new gravity-assisted guidance system with a standard mechanical guide. Three hundred ten cups were impacted by 5 surgeons, and the final cup orientation was measured. With the new guide, the average error in anteversion was 0.4 degrees , compared with 10.4 degrees with the standard guide and 0.3 degrees and -4.7 degrees , respectively, for abduction angles. The average time required for orienting the cups was similar for both guides. The accuracy and reproducibility obtained with the new guide were better (P < .0001). These good results would require a clinical validation.

Acetabulum↗