Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.263169
Source of Support: None, Conflict of Interest: None
Keywords: Microgravity analogue, microgravity, neuroglia, neurology, sensorimotor
As life evolved on Earth, a myriad of changes in the physical and chemical factors invoked adaptations and selections that induced the convoluted evolutionary pressures. Earth's gravitational force has been constant in direction and magnitude since the formation of the planet., All living beings including animals, plants and humans, for this reason, have advanced to contrive with and rely upon gravity. From time immemorial, all living organisms adapted their cellular and operating functions to gravity characteristic of our planet Earth.
Simulated microgravity is established on the premise that absence of weight load is realized, similarly to nullification of sedimentation during weightlessness (0 g). In simulated microgravity circumstances, complete weightlessness is never entirely reached, as weak residual acceleration forces are still active. Ground-based devices such as the random positioning machine, the rotating wall vessel and the fast-rotating clinostats have successfully replicated microgravity responses. However, these devices generally seem to underestimate the effects observed in real microgravity during spaceflights. Altered gravity conditions like hypo- and hyper- gravity have had differential effects on living beings at various levels of organization, ranging from changes in the biophysical properties of single cells to the intact nervous system.
It is important to incorporate the different findings into a consistent model to gain insights into how the components of the nervous system interact as a response to the short- and long-term gravitational variation. With emphasis on future space travel, including space tourism, planned long-term manned missions to Moon, Mars and interplanetary explorations, it is imperative to understand the impact of altered gravity on the nervous system and the gravity-perceiving systems, which determines movement, cognition, and survival.
Studies of astronauts participating in space missions on the International Space Station (ISS) have demonstrated spaceflight-induced ocular changes such as choroidal folds, optic disk edema, globe flattening, and hyperoptic shifts. In vitro studies on different types of human renal cortical cells or mouse MC3T3 osteoblasts, hFOB human fetal osteoblasts, in space or on microgravity simulating devices, have demonstrated significant changes in gene expression patterns,,, increased apoptosis [ML1 follicular thyroid cancer cells, glial cells, MDA-MB231 breast cancer cells and human lymphocytes (Jurkat)],,, and induction of autophagy (human umbilical vein endothelial cells, HEK293 cells),, as well as changes in differentiation (FTC-133 follicular thyroid cancer cells), migration, cell adhesion, extracellular matrix composition (ML1 cells) and alterations in the cytoskeleton (FTC-133 cells, A431 epidermoid carcinoma cells)., The nervous system controls muscle contraction permitting the body to withstand the gravitational force and manage locomotor patterns and reflexes during the evolutionary shift from aqueous to terrestrial life. One of the fundamental circuits within the central nervous system (CNS) to control muscle contraction is the monosynaptic reflex arch. The monosynaptic reflex arc, which is an important circuit within the central nervous system, responds to an external stimulus, which leads to fast muscle contractions. The measure of muscle contraction depends on the size of the sensory input.
In order to maintain a simple posture or movement for mobility of the terrestrial life, the nervous system processes sensory information from the vestibular, visual systems and proprioception. These sensorimotor competencies are crucial for life.,,,, Further, there are modifications in the neuromuscular system underlying health-related changes that open up many questions on how the variation of gravity influences the nervous system. Countermeasures to re-establish homeostatic states within the human body have begun, but the pathophysiologic mechanisms of altered gravity on the neurologic system are still obscure. Here, we recapitulate the current knowledge of neurologic adjustments encountered or expected from space exposure, the most unconventional environmental pressure to affect the human species.
The direct effects of microgravity on various tissues of the musculoskeletal, cardiopulmonary, immune, nervous and vestibular systems are well documented. Nevertheless, at a cellular level, these effects are largely indirect and are moderated through the modification of a complex variety of physicochemical affects, such as hemodynamic and hydrostatic pressure, fluid shear stress, three-dimensional tissue stress, mass transport, and permeability. These direct and indirect effects can be sensed by cells in many ways, for example, via membrane-bound receptors and ion channels, primary cilia, cell shape, cytoskeletal and membrane structure changes. Cell's morphological polarity determines its behavior. For instance, non-polarized cells are more likely to undergo apoptosis., Depending on the origin of cells, apoptosis may be increased or decreased and it may be affected when the cell cytoskeleton is disorganized. An increase in cell apoptosis is a significant consequence of the changes in cell structure and function that occur in microgravity. Microgravity has effects on both cell shape and cytoskeleton.
For long, it has been assumed that cells are too insignificant in mass to be affected by microgravity. But in reality, space travel, and in particular microgravity, does affect cells. Multiple experiments from the past three decades have demonstrated that spaceflight significantly changes the morphology and function of human and microbial cells in culture.,,,,,,,, The history of gravitational cell biology can be traced back to the nineteenth century, when Julius Sachs (1863), Charles Darwin (1880), and Wilhelm Pfeiffer (1904) investigated the influence of gravity on plants, with the downward orientation of root caps. In the 1960s, early satellites had some bacterial, plant, and animal experiments aboard. However, these experiments had logistical and technical constrains which were overcome by the Skylab platform that allowed scientists to embark on a new era in microgravity research.
These early studies set the stage for investigation of the effects of microgravity on red blood cell shape, bone loss, muscle deconditioning, metabolic changes, and immune performance. Following the Skylab based research, the Shuttle era opened new vistas for scientific experiments. It was during this time, and in the follow-up studies on the Shuttle program that some of the key findings with regard to mammalian cells in space took place. These findings include: (1) Inhibition of lymphocyte activation, cytokine production, and locomotion;,, (2) interference with transmembrane signaling;, (3) changes in metabolism;,, and (4) tissue morphogenesis.,
Our experimental methods for investigating the effects of microgravity on biological systems are limited. Space is the most reliable environment in which to conduct these experiments; however, there is currently limited access to the space-flight research platform. In the absence of routine spaceflight opportunities, there are several approaches that provide brief periods of analogue conditions; namely, drop towers, parabolic flights, suborbital missions, supported by ground-based model systems for simulated microgravity such as clinorotation, rotating wall vessels, three-dimensional (3D)-random positioning machines and diamagnetic levitation. [Figure 1].
The nervous system is the master controlling and communicating system of the body. It is a network of neurons and fibers which transmits nerve impulses between parts of the body. Cells communicate via electrical and chemical signals. The nervous system originates from the neural tube and neural crest, formed from the ectoderm. The neural tube becomes the central nervous system. Neuroepithelial cells of the neural tube proliferate into a number of cells needed for development.
Neuroblasts become amitotic and migrate, and later sprout axons to connect with targets and become neurons. The neuronal plasticity of the adult brain depends on the correct timing and expression of primordial cell proliferation and migration, before entering into their differentiation processes. The gravity on earth is responsible for maintaining a conditioning fluctuation state of cells and their organelles, which is lost in weightless conditions.
The Neurolab flight was the third space shuttle mission dedicated to life science research, but it was the first mission to focus distinctly on how the neurological system responds to the challenges of space flight. During the early years of the National Aeronautics and Space Administration's (NASA's) manned space flight program, efforts in the life sciences were driven by the operational medicine and biomedical support of short duration ventures such as the Mercury and Gemini missions. During these flights, according to the NASA literature, no significant problems arose regarding sensory system function. However, during the Apollo missions, a number of astronauts reported mild-to-severe motion sickness, and in the early 1970's, NASA initiated studies of the illness. More recently, attention has been devoted to other physiological and behavioral changes that occur as a result of space flight. The data obtained throughout the Neurolab project starting in 1990's till 2003, showed that the absence of gravity had limited, and reversible, effects on the functional capabilities of the mature human brain.
The Neurolab experiments have also affirmed that microgravity influences not only the sensory input, but also the brain integrating functions, and consequently its motor output. In the microgravity environment, 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. The 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, the 30-day flight experiments performed on the Russian Bion-M1 biosatellite, using male mice, 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 inadaptation to Earth's gravity. Moreover, the expression of genes involved in dopamine synthesis and degradation was reduced, although this did not modify the expression of the main genes of serotonin metabolism and signaling. It did, however, reduced 5 hydroxy-tryptamine 2A (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.
Even though there is ample evidence supporting the hypothesis that exposure to microgravity has contrasting effects on the nervous system, in some cases it has been shown that microgravity does not disturb 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.,, A three-dimensional (3D) in-vitro neuroglial co-culture model was proposed by Morabito and co-workers (2015), using GL15 and SH-SY5Y cells, which are astrocyte-like and neuronal-like cells, respectively. The cells grown as co-culture under simulated microgravity, as compared to cells grown as static monolayers, showed a significant increase in the expression of some differentiation-specific markers, such as glial fibrillary acidic protein (GFAP) and S100B in the glial cells, and growth associated protein 43 (GAP43) in the pseudo-neurons. Also, both of these cell types showed an increased expression of neural cell adhesion molecule (N-CAM) and conexin 43 (Cx43), as a result of increased functional cell-cell interactions.
Studies of astronauts participating in missions on the International Space Station have shown prominent adverse outcomes in visual function. The adverse outcomes were observed in astronauts after a long-term spaceflight and it has been hypothesized that these visual changes are connected to cephalad fluid shifts, intracranial pressure and optic nerve sheath compartment syndrome, as a consequence of prolonged microgravity exposure.,, Simulated microgravity induces significant alterations in the F-actin-cytoskeleton and cytoskeleton-related proteins of human retinal pigment epithelial cell line (ARPE-19), in addition to changes in the cell growth behavior and gene expression patterns involved in cell structure, growth, shape, migration, adhesion and angiogenesis.
The cell membrane lipid bilayer properties are known to respond to various changing conditions such as temperature, deformation, pH, specific ions, and gravity. Also, changes are seen in membrane structure, composition, bi-leaflet organization, lipid rafts, and in association with the cytoskeleton [Figure 2]. These changes are presumed to be due to the initial response of the cell, minimizing its surface area-to-volume ratio. From experiments with unicellular organisms  and various cell types, such as the immune cells  and the neuronal cells, it is well established that single cells react to changes in gravity even though they do not have dedicated gravity-sensing structures. Neurons can change their membrane potential in order to communicate. This ability is based on the activity of the integrated membrane proteins as ion channels and ion pumps. The physicochemical state of the lipid membrane can directly modify the function of membrane proteins., In experiments conducted in the non-microgravity environment (1g), it was shown that with decrease in membrane fluidity, the closed-state probability of nicotinic acetylcholine receptors (nAChRs) increased.
These nAChRs are major players in the sensorimotor system as they are located in the motor end plates that form the interface between the neuronal system and the muscles. Experiments performed in micro- and hyper-gravity models using artificial asolectin vesicles and human neuronal SH-SY5Y cells showed a significant increase in the membrane fluidity in microgravity and its decrease in hyper-gravity.
Historically, we have known about mechanosensitive channels and their ability to inform the cells of the environment with respect to hydrodynamic/fluid shear, physical impact, stretching, and perhaps even microgravity itself. These channels are responsible for modulation of ion flow in and out of the cell, and as a consequence, can modulate a number of cascading activities beyond that., Ion channels are pore forming proteins that allow ions to pass through the channel pore. These ion channels are crucial for neuronal communication. Neuronal ion channel activation occurs in either a voltage-gated or a ligand-gated manner. Ion channels play crucial roles in neuronal signal processing ,, and plasticity.,, The physiological function of many ion channels that have been identified to be mechanosensitive is still unknown.
Thus far, in microgravity research, no native ion channel proteins have been used. Instead, pore-forming peptides, which can be used as ion channel analogs, have been employed to study the ion channel parameters as open- and closed-state probability. Microgravity experiments have showed that the open-state probability of porin channels from Escherichia More Details coli is significantly decreased, whereas in hyper-gravity, it is increased. Additionally, alamethicin, an artificial pore-forming peptide from Trichodermaviride, manifested decreased activity in microgravity and hyper-gravity., Moreover, in parabolic flight experiments, native oocytes from Xenopus laevis, as well as oocytes that overexpress epithelial sodium channels (ENaC), demonstrated reduced membrane conductivity under microgravity and increased conductivity under hyper- gravity., With the onset of a different gravity condition, the open-state probability is changed, returning to normal as soon as the experiment returns to the normal 1 g gravity. Gravity influences the velocity of propagating waves in the excitable media of neuronal tissue. Other experiments have shown that electrical properties of neurons are not affected by microgravity, and cell characterization by immunostaining shows a normal neuronal phenotype. In contrast to these findings, Ranjan and colleagues (2014) observed that in rats exposed to simulated microgravity, some areas of the active zone of the cornu Ammonis 1(CA1) hippocampal neurons significantly decreased, while dendritic arborization and the number of spines significantly increased. A stable resting membrane potential is essential for neurons to communicate, and this can be modulated by changing the activity of relevant ion channels. Experiments on the parabolic flight during microgravity phase have shown significant depolarization (3mV) of the resting potential of human neuronal cells, and vice versa, in hyper-gravity. Another microgravity analogue model obtained by drop tower, showed that the rate of action potentials triggered by the spontaneously active leech neurons is significantly increased. The action potential kinetics are gravity dependent as well. The action potential propagation velocity on the axonal level is decreased in microgravity and is increased in hyper-gravity, which was demonstrated in parabolic flight missions in vitro in isolated earthworm axons and isolated rat axons, and in vivo in intact earthworms. All of these changes are fast and fully reversible.
Microgravity provides environmental characteristics that are difficult to obtain on Earth. Using extreme conditions that modify the physical force environment to investigate life processes affords opportunities for the discovery and development of applications that significantly enhance research. 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.
Till date, many in vitro and in vivo experiments have been conducted to investigate the effects of micro- and hyper-gravity on neuronal tissue. From molecular-to-cellular-to-system levels (both neuromuscular and nervous), all have shown to adapt to changes in the gravity conditions. The new International Space Station strategy affords multiple experiments and real-time modifications. In combination with the ongoing improvements in flight hardware that incorporate experimental flexibility, modularity, and advanced capabilities to enable a broad range of biological experiments, these experiments are enhancing the potential for innovative biomedical and biotechnological applications. Additionally, valuable microgravity analogues can be used on the ground to profile experiments for spaceflight to identify specific biological characteristics to be observed in microgravity. Countermeasures such as vibration treatment, sensorimotor training, and artificial gravity have been applied to counter the deleterious effects of changes in gravity.
Microgravity, thus, affords a new window through which one may observe life processes. In the short time that we have studied microgravity, it is apparent that terrestrial life responds to the physical change in gravity by altering gene expression, inhibiting cellular movement, promoting differentiation, and inducing profound morphological changes. Cells adapt, and most survive the transition. In experiments conducted over a short duration in the microgravity environment, the changes are genuinely phenotypical, and therefore, resolve when the gravity status is returned to 1g. Microgravity is thus a probe which, as with other physical stresses, can reveal novel mechanisms that are fundamental to the cell processes, disease processes, and the adaptation of living systems to changes in physical forces. As astronauts travel to Mars and deeper outer space, they will live in the absence of gravity for extended periods of time with transition between weightlessness, isolation, outer space radiation and altered planetary gravitational forces during various phases of the mission. Considering the gravity dependency of the nervous system, further research and countermeasure developments will be necessary to assure a safe space travel and Earth return in the future.
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[Figure 1], [Figure 2]