The Effects of Simulated Microgravity on the Human Nervous System: the Proposal of a Three-dimensional Glia–neuron Co-culture Cell Model

Over the last few decades, the increasing number of spaceflights and the permanence of astronauts in orbit to maintain satellites and space stations have focused attention on the effects induced by altered gravitation on the human body, and in particular on bone, skeletal muscle, and brain function. The study of the mechanisms that underlie the effects induced by microgravity and the search for protection strategies are made difficult by limitations due to logistic difficulties (such as the operators and/or automatic processes of measurement during flights) and limitations in the gathering of enough material from astronauts. This has prompted the need to develop and improve alternative models of a simulated weightless environment that can be used to overcome these critical phases. In addition, the use of in-vitro cell models can allow in-depth studies of cellular targets of the extracellular mechanical forces that are modified by variations in gravity. This overview highlights some aspects of the research in this field that are focused on the effects of microgravity on nerve cells, whereby their modified functions might be related to sensory/motor impairment as well as to the disorders of mood, behavior and cognition experienced after spaceflights. To cite this article: Maria A. Mariggiò, et al. The effects of simulated microgravity on the human nervous system: The proposal of a three-dimensional glia–neuron co-culture cell model. During nervous system development, the primordial cells proliferate and migrate to the positions they will be in the adult brain before entering into their differentiation processes. The correct timing and expression of these events involve both cellular interactions and the interplay of organelles within the cells themselves. These aspects are also the main determinants of neuronal plasticity in the adult brain. The physical environment of the cells and their organelles can influence these interactions. Indeed, on Earth, gravity is responsible for maintaining a conditioning fluctuation state of cells and their organelles, while in the weightlessness of space these differences will be lost [1]. The Neurolab project was developed during the 1990's, and it represented the first attempt for a comprehensive approach to study the effects of microgravity on a specific field of research: the brain and the nervous system. In 2003 [1] the main results from a 16-day space mission were collected and published, these highlighted an international program of 26 experimental projects in the neurosciences that had been selected from among 172 proposals from scientists worldwide. Although the …

During nervous system development, the primordial cells proliferate and migrate to the positions they will be in the adult brain before entering into their differentiation processes.The correct timing and expression of these events involve both cellular interactions and the interplay of organelles within the cells themselves.These aspects are also the main determinants of neuronal plasticity in the adult brain.The physical environment of the cells and their organelles can influence these interactions.Indeed, on Earth, gravity is responsible for maintaining a conditioning fluctuation state of cells and their organelles, while in the weightlessness of space these differences will be lost [1] .
The Neurolab project was developed during the 1990's, and it represented the first attempt for a comprehensive approach to study the effects of microgravity on a specific field of research: the brain and the nervous system.In 2003 [1] the main results from a 16-day space mission were collected and published, these highlighted an international program of 26 experimental projects in the neurosciences that had been selected from among 172 proposals from scientists worldwide.Although the peculiar logistic situation and the limited time duration (about 2 weeks) of the test phase must be considered, much of the data obtained showed that the absence of gravity had limited, and reversible, effects on the functional capabilities of the mature human brain [1] .

RESEARCH HIGHLIGHT
This scenario is quite different, and in some aspects more complex and dynamic, for data obtained from the study of the changes induced by microgravity during the development of the nervous system and/or during some activities that stimulate the plastic capacity of the mature brain.A condition of weightlessness affects the whole sensory system, and primarily the vestibular functions.The Neurolab experiments have provided evidence of adaptation of the vestibular system to microgravity, according to sensations experienced by spaceflight crewmembers upon their return to Earth, and to changes from gene expression to the output of the vestibular system that have occurred within the brain itself in animal models that have been carried out as part of the Neurolab mission [1] .In addition, microgravity influences not only the sensory input, but also the brain integrating functions, and consequently its motor output.Indeed, in space, the balance between the senses needs to be re-organized; e.g., the limbs are weightless in space, and this implies that some movements have to be re-calibrated [1] .
Neurolab experiments performed on animal models during their development have suggested that microgravity affects nervous system development, to induce long-lasting modifications, compared to this development on Earth.Also, depending on the animal species, there are critical periods when gravity is of particular importance to the developmental process.
More recently, a new international biomedicine research program was carried out in space during the 30-day flight of the Russian Bion-M1 biosatellite (April 19 to May 19, 2013).This satellite was an automated spacecraft that was dedicated to biomedical experiments [2] .The space experiments were performed using male mice, and they revealed that after the return from the 30-day spaceflight, the mice were markedly less active compared to the control groups that had remained on Earth, and they displayed signs of pronounced disadaptation to Earth gravity [2] .Interesting data have come from the analysis of gene expression of pro-apoptotic and anti-apoptotic factors in different brain regions of these mice after their 30-day spaceflight.The spaceflight failed to alter the expression of the pro-apoptotic BAX gene in all of the brain structures analyzed, as well as the expression of the trophic factor BDNF and its receptors.On the other hand, after long-term spaceflight, expression of the anti-apoptotic BCL-XL gene was reduced in the striatum and hypothalamus, while it was increased in the hippocampus.Thus, it has been hypothesized that the imbalance of the expression of these pro-apoptotic and anti-apoptotic factors probably underlies the development of the behavioral abnormalities after long-term spaceflight [3] .At the same time, the increase in BCL-XL in the hippocampus might have a compensatory role, to counteract the harmful effect of long-term spaceflight on brain performances [3] .
Another part of the puzzle was revealed from analysis of brain genes that control the dopamine and serotonin systems.The one month stay in space of these mice decreased the expression of genes involved in dopamine synthesis and degradation, although this did not modify the expression of the main genes of serotonin metabolism and signaling.It did, however, reduced 5-HT2A receptor gene expression in the hypothalamus.For these reasons, these alterations to the dopamine system might also contribute to spaceflight-induced neuromotor impairment that has been detected in both humans and mice [4] .
However, although there have been many studies on the effects on the nervous system of absence of gravity during spaceflights, these have been heterogeneous, fragmentary, and far from conclusive, especially those concerning the process of maturation and growth of the nervous system.In particular, apart from the expected logistic difficulties (also in terms of the limitations of the operators and/or the automatic processes of measurement), the main limitation has been the short exposure times, which have only reached up to 30 days.To carefully measure the changes induced by the absence of gravity during the phases of growth and maturation of the nervous system, longer exposure periods are needed.This requires the need to develop and improve alternative models of a weightless environment to overcome the critical phases just outlined.
A second equally important aspect to be considered concerns the modality by which experiments are carried out in orbiting stations and/or during spaceflights.Under these conditions, even if it is preeminent, the gravity decrease is not the only environmental change in comparison to what happens in ground experiments.Indeed, to be sure that the modifications observed are the consequence of the lack of gravity, it would be necessary to also have some controls on a flight that are subjected to an artificially created gravitational force.These two situations for the creation on the Earth of a simulated microgravity environment and the presence on spaceflights of artificial gravity have been the subjects of studies and technological efforts by different international teams involved in aerospace research.In addition, starting from the 1990's, many efforts were made to solve the problem of growth of cell cultures or tissue explants under three-dimensional (3D) conditions, while also investigating the presence of reduced gravity.Attempts have been directed toward the design of an apparatus (known as a bioreactor) that can be used to modify in-vitro environments to imitate those present in situ.This would thus allow the study of interactions that occur between the tissue microenvironments, the proteins of the extracellular matrix, and the metabolism within the cell also during the exposure of such cells to the microgravity conditions of spaceflight.
Briefly, a bioreactor uses mechanical resources to influence biological processes.Bioreactors can contribute to in-vitro formation of tissue-like cell aggregates, and they can also regulate the extracellular signals received by cells.Bioreactors allow the delivery of the required nutrients and gases to tissue cultures.In addition, they facilitate nutrient transport to cells and waste transport from tissues.Bioreactors, and especially microgravity bioreactors, can maintain a spatially uniform cell distribution throughout the tissue-engineering scaffold [5] .Some studies have already shown that different mechanical stimuli can play critical roles in cellular shape, and in tissue geometry and function, such as fluid-flow shear stress, hydrostatic pressure, substrate strain deformation, and gravity [6, 7]   .Microgravity promotes co-location of cells and the initiation of differentiative cellular signaling via induction of specialized cell-adhesion molecules and extracellular matrix proteins.Microgravity has also been shown to potentiate stem-cell proliferation while sustaining their ability to differentiate, which can be of great importance in tissue engineering [8] .
A device was developed in the NASA laboratories, and it is based on the principle of the clinostat, which is a device that can be used for the cultivation of cells both on the Earth and in space [9,10] .This apparatus is a rotating bioreactor in the form of a monoaxial clinostat (a rotary cell-culture system [RCCS] bioreactor), as a horizontally rotating and fluid-filled culture vessel.This is equipped with a gas-exchange membrane that optimizes the oxygen supply to the biological samples.Without air bubbles or an air-liquid interface, the fluid dynamics conditions inside the culture chamber generate a laminar flow state, which greatly reduces shear stress and turbulence, processes that are hazardous for cell survival (Fig. 1).When the gravitational forces are balanced with the centrifugal forces, microgravity-like culture conditions are created within the cylinders in the annular space.When the aggregates of cultured cells grow in size, the speed of rotation of the culture vessel can be increased to compensate for their increased sedimentation rate [11,12] .
Recent studies have shown that microgravity affects the functioning of the nervous system, although the possible physiological mechanisms of these effects remain difficult to define [13,14] .These difficulties are mainly due to the poor models that are available, which arises either because of their high cost and low availability (e.g., spaceflight), or because they are little representative of true microgravity conditions (e.g., hindlimb suspension/disuse model).Among the ground-based models, in-vitro cultures of cells/tissues within clinorotation-based systems (e.g., random positioning machine, RCCS bioreactors) represent a reasonable alternative to spaceflight.
The RCCS bioreactor, in particular, was initially developed by NASA engineers to maintain cells in culture during space missions and to counteract the forces faced during shuttle launch and landing.The RCCS bioreactor has also been used to maintain cells in dynamic 3D culture on the ground, and because of its particular properties, the RCCS bioreactor also allows the modeling of microgravity on the ground.Setting standardized parameters, it is possible to promote the co-localisation of cells, the establishment of cell-cell contacts, and consequently, the spontaneous formation of multicellular aggregates [11,12,15] .Moreover, the rotation speed can be regulated in such a way that it is possible to reach a vector-averaged gravity that simulates low-gravity conditions [16] .
Although a wealth of evidence supports the hypothesis that exposure to microgravity has contrasting effects on the nervous system, in some cases it has been shown that microgravity does not perturb cell differentiation and tissue assembly.In other cases, microgravity appears to strongly alter cell morphology and function, or to even improve stem cell differentiation into neurons [17] .Nevertheless, few studies have examined whether microgravity affects the development of neurons in culture.In studies using suspensions of dissociated cortical neuronal cells from rat embryos, 24h exposure of simulated microgravity did not affect the ability of neural cells to develop normally and to differentiate.Also electrophysiological recordings have indicated that the electrical properties of neurons are not affected by microgravity, and cell characterization by immunostaining shows a normal neuronal phenotype.Treatment in simulated microgravity has revealed only increases in glial fibrillary acidic protein fluorescence in elongated stellate glial cells [13] .In contrast with these findings, Ranjan and colleagues (2014) observed that in rats exposed to simulated microgravity, some areas of the active zone of CA1 hippocampal neurons significantly decreased, while dendritic arborization and the number of spines significantly increased [18] .
As indicated above, the effects induced by modifications to gravity on neuronal behavior have been investigated under microgravity conditions in environments such as spatial vectors or in bioreactors, where the gravity was reduced artificially.In contrast, cell culture experiments (other than neuronal cells or tissue) have been performed mainly on osteoblast or osteoclast cells under hypergravity conditions, and have been described recently.Exposure to hypergravity has effects on the whole cell mass, and cells exposed to 2× or 3×g reduce their height by 30% to 50% on average, showing a decrease in the height of their microtubule network, and also an increase in the thickness of actin fibers, with no effects on cell viability [19] .These effects on osteoblast morphology and differentiation under continuous increased gravity over longer time periods have shown the induction of favorable effects on genes involved in osteoblastogenesis [7] .
For the application of hypergravity to cultured neurons or neuronal-like cells, such as those derived from tumors of nervous origin (e.g., PC-12, SH-SY5Y, GL15 cells), there remains little or no data.After a critical review of the literature available, only one significant study on the effects of increased gravity in PC12 cells can be found [17] .In this study, the influence of hypergravity on these neuron-like cells was analyzed 48 h after cell incubation under increased gravity (at 50× and 150×g, for 1 h) to determine the effects of this treatment on cell proliferation and differentiation mechanisms.These data show that the development of the neuronal phenotype is strongly affected by hypergravity treatment, as the neurites in the treated samples were significantly longer compared to those in the control cultures.Under these conditions, hypergravity increased the transcriptional patterns of genes involved in neuronal maturation in a dose-dependent manner: the neurofilament-66 and 3-tubulin genes.
The study of brain maturation processes both from a cellular point of view and as an integrative scenario is also difficult, considering the peculiar tissue organization of this fundamental organ.The structure and functions of the different parts of the nervous system depend on the close relationships that are activated in the first phases of tissue growth, between two cell types: neurons and glial cells.These cells continue to influence each other without losing their own peculiarities.Indeed, neurons and glial cells represent two sides of the same coin, as two partners within the same functional structures, and their relationships are so tight that nothing can happen in neurons themselves and to the neuronal network without affecting the glia (and the microglia especially), and vice versa.
Neurons are responsible for the conduction and transmission of nerve impulses, and glial cells are not only implicated in structural, nutritional and immune support to these excitable cells, but are also involved in the elaboration of information, as are the neurons.For this reason, the study of the effects of environmental changes (whatever these changes might be) in the nervous system that affect cell shape, the extracellular matrix, extracellular space, and intercellular interactions should always be analyzed according to their impact on both of these two cell types that constitute it.
Recent progress in this field has included the availability of different types of expanded cells from cultures of neuronal and glial cells.These cell models have provided significant advances in the understanding of some of the mechanisms of diseases of the nervous system, although to date, the full characterization of in-vitro models is only partially accepted by the international research field.Consensus is still lacking on the methodological approaches, so it is still necessary to validate such strategies and techniques that allow accurate adaptation to new genetic or environmental situations [20,21] .These considerations formed the basis of a recent study of Morabito and colleagues (2015) that can be considered the first organic study carried out in artificial microgravity.This study investigated the early stages of development of nerve tissue using co-cultures of glial cells and neurons.Here, Morabito and co-workers (2015) proposed a 3D in-vitro neuroglial co-culture model to evaluate the ability of these cells to reproduce neuronal features, at least in part.For this, they used two well-characterized human cell lines, GL15 and SH-SY5Y cells, which are astrocyte-like and neuronal-like cells, respectively.The data from this study show that compared to cells grown as static monolayers, co-cultures of these neuronal-like and glial-like cells under artificial microgravity showed significant increases in the expression of some differentiation-specific markers, such as GFAP and S100B in the glial cells, and GAP43 in the pseudo-neurons.In addition, both of these cell types were positively modulated for their expression of N-CAM and Cx43, as a result of increased functional cell-cell interactions [22] .With the purpose of this study being to develop a 3D, dynamic, in-vitro neuroglial co-culture system to evaluate the ability of these cells to reproduce some specific aspects of the nervous system, this can be considered to have been sufficiently demonstrated.This provides the way to propose this cell model in the scientific scenario as a bridge between the classical 2D in-vitro systems and animal models for the study of the growth and/or maturation of the nervous system.In addition, the importance of this scheme is also evident considering that cell-cell interactions between glial cells and neurons are crucial not only for both glial and neuronal differentiation and developmental processes, but also for response to neural injury [23] .
In conclusion, taking into consideration what has been discussed above, the 3D co-culture of neuronal and glial cells in rotating bioreactors can provide in-vitro models for physiological and pathophysiological investigations also in still unexplored fields, such as the development of the nervous system.A general problem that remains open is to determine any genetic alterations and/or functional failures in cells and tissues grown under microgravity, as these might adversely affect patients treated with these products.Studies and progress in this field remain very promising, and there is a real hope that in the foreseeable future replacement therapies for defective tissues will be a viable option.

Figure 1 .
Figure 1.Schematic diagram of a bioreactor.The arrows indicate the direction of rotation.