Experiments performed in orbit on the central nervous system have focused on the control of posture, eye movements, spatial orientation, as well as cognitive processes, such as three-dimensional visual perception and mental representation of space. Brain activity has also been recorded during and immediately after space flight for evaluating the changes in brain structure activation during tasks involving perception, attention, memory, decision, and action. Recent ground-based studies brought evidence that the inputs from the neurovestibular system also participate in orthostatic intolerance. It is, therefore, important to revisit the flight data of neuroscience studies in the light of new models of integrative physiology. The outcomes of this exercise will increase our knowledge on the adaptation of body functions to changing gravitational environment, vestibular disorders, aging, and our approach towards more effective countermeasures during human space flight and planetary exploration.
Research Containing: Humans
In our previous studies, we have shown that the occurrence of geometric illusions was reduced in vestibular patients who presented signs of otolith disorders and when healthy observers were tilted relative to gravity. We hypothesized that the alteration in the gravitational (otolith) input was responsible for this change, presumably because of a connection between vestibular and visual-spatial cognitive functions. In this study, we repeated similar experiments in astronauts during long-duration spaceflight. In agreement with the data of otolithic patients, the inverted-T geometric illusion was less present in the astronauts in 0 g than in 1g. In addition, the vertical length of drawings made by astronauts in orbit was shorter than that on the ground. This result is also comparable with the otolithic patients who perceived the vertical length of line drawings to be smaller than healthy individuals. We conclude that the impairment in the processing of gravitational input in long-duration astronauts affects their mental representation of the vertical dimension similar to the otolithic patients. The astronauts, however, recover to baseline levels within 1 week after returning to Earth.
Constant velocity off-vertical axis rotation (OVAR) provides dynamic linear acceleration stimuli that can be used to assess otolith function. Eight astronauts were rotated in darkness about their longitudinal axis 20 degrees off vertical at low (0.125 Hz) and high (0.5 Hz) frequencies and their responses were compared before and after spaceflight. Eye movements were recorded using infrared videography and perceived motion was evaluated using a joystick with four degrees of freedom – pitch and roll tilt, front-back and lateral translation. Low-frequency OVAR generates tilt otolith-induced responses – modulation of ocular counter-roll and counter-pitch with perceived conical motion path – whereas high-frequency OVAR generates translational otolith-induced responses – modulation of horizontal and vergence slow phase velocity with perceived cylindrical motion path. While there were transient changes in the amplitude of the translational ocular responses on landing day, there were no major changes in the tilt ocular reflexes after adaptation to weightlessness. However, there was an increase in sensitivity to motion perception after spaceflight. Direct comparisons of pre- and postflight stimuli suggested that OVAR on landing day was less provocative of motion sickness than before spaceflight. These results confirm that some otolith reflexes elicited during passive motion may not be altered by short-duration spaceflight – or may readapt very quickly – and that the resolution of sensory conflict associated with postflight recovery involves higher-order neural processes.
Validation of centrifugation as a countermeasure for otolith deconditioning during spaceflight: preliminary data of the ESA SPIN study
In the framework of further space exploration, countermeasures to combat the drawbacks of human space flights are essential. The present study focuses on the influence of microgravity on the otolith-ocular reflex and aims to test the hypothesis of artificial gravity being an adequate countermeasure for the deconditioning of the aforementioned reflex. The so-called SPIN study, commissioned by the European Space Agency, can be considered as a control experiment in the broad sense for the Neurolab mission (STS-90) during which 4 crewmembers of the space shuttle were subjected to in-flight centrifugation on the visual and vestibular investigation system (VVIS). After their nearly 16-day mission, they did not suffer from orthostatic intolerance and spatial disorientation. In addition, the relevant parameters of the otolith-ocular interaction remained unaffected. For this study cosmonauts from a long duration stay in the International Space Station that were not centrifuged in-flight were tested on the VVIS (1 g centripetal interaural acceleration) on 6 different days. Three measurements were taken about 1.5-2 months prior to launch and 3 were taken at 1, 4 and 9 days after return from space. Ocular counter-rolling was measured before, during and after rotation on the VVIS using infrared video goggles and compared pair wise using Friedman tests. The perception of verticality was monitored using an ultrasound system for perceptual evaluation. The preliminary results of 4 cosmonauts showed a surprisingly large inter-individual variability of the measurements. Although OCR and perception of verticality appeared to be influenced overall by the exposure to microgravity, the wide variability among the cosmonauts obscured any statistical significance, in particular due to one cosmonauts being inconsistent with the other 3. Despite the specificity of the tests under normal conditions, the diverse response to spaceflight of our subjects exposes the complexity of the peripheral and central neural adaptive processes.
Effects of pleiotrophin overexpression on mouse skeletal muscles in normal loading and in actual and simulated microgravity
Pleiotrophin (PTN) is a widespread cytokine involved in bone formation, neurite outgrowth, and angiogenesis. In skeletal muscle, PTN is upregulated during myogenesis, post-synaptic induction, and regeneration after crushing, but little is known regarding its effects on muscle function. Here, we describe the effects of PTN on the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles in mice over-expressing PTN under the control of a bone promoter. The mice were maintained in normal loading or disuse condition, induced by hindlimb unloading (HU) for 14 days. Effects of exposition to near-zero gravity during a 3-months spaceflight (SF) into the Mice Drawer System are also reported. In normal loading, PTN overexpression had no effect on muscle fiber cross-sectional area, but shifted soleus muscle toward a slower phenotype, as shown by an increased number of oxidative type 1 fibers, and increased gene expression of cytochrome c oxidase subunit IV and citrate synthase. The cytokine increased soleus and EDL capillary-to-fiber ratio. PTN overexpression did not prevent soleus muscle atrophy, slow-to-fast transition, and capillary regression induced by SF and HU. Nevertheless, PTN exerted various effects on sarcolemma ion channel expression/function and resting cytosolic Ca(2+) concentration in soleus and EDL muscles, in normal loading and after HU. In conclusion, the results show very similar effects of HU and SF on mouse soleus muscle, including activation of specific gene programs. The EDL muscle is able to counterbalance this latter, probably by activating compensatory mechanisms. The numerous effects of PTN on muscle gene expression and functional parameters demonstrate the sensitivity of muscle fibers to the cytokine. Although little benefit was found in HU muscle disuse, PTN may emerge useful in various muscle diseases, because it exerts synergetic actions on muscle fibers and vessels, which could enforce oxidative metabolism and ameliorate muscle performance.
Formation and structure of transplantable tissue constructs generated in simulated microgravity from sertoli cells and neuron precursors
Cell transplantation therapy for Parkinson's disease (PD) has received much attention as a potential treatment protocol for this neurodegenerative condition. Although there have been promising successes with this approach, it remains problematic, especially regarding the inability to provide immediate trophic support to the newly grafted cells and the inability to prevent acute and/or long-term graft rejection by the host. To address these issues of cell graftability, we have created a novel tissue construct from isolated rat Sertoli cells (SC) and the NTerra-2 immortalized human neuron precursor cell line (NT2) utilizing NASA-developed simulated microgravity technology. The two cell types were cocultured at a 1:4 (SC/NT2) ratio in the High Aspect Rotating Vessel (HARV) biochamber for 3 days, after which a disc-shaped aggregate (1-4 mm diameter) was formed. Sertoli neuron aggregated cells (SNAC) were collected by gravity sedimentation and processed either for light and electron microscopy or for fluorescent immunocytochemistry. Intra-SNAC clusters of SC and NT2 cells were identified by anti-human mitochondrial protein (huMT–specific for NT2 cells) and cholera toxin subunit B (CTb–specific for SC). There was little evidence of cell death throughout the aggregate and the absence of central necrosis, as might be expected in such a large aggregate in vitro. Ultrastructurally, SC did not express junctional modifications with NT2 cells nor with adjacent SC as is typical of SC in vivo and, in some protocols, in vitro. NT2 cells, however, showed distinct intercellular junction-like densities with adjacent NT2 cells, often defining canaliculi-like channels between the microvillus borders of the cells. The results show that the use of simulated microgravity coculture provides a culture environment suitable for the formation of a unique and viable Sertoli-NT2 (i.e., SNAC) tissue construct displaying intra-aggregate cellular organization. The structural integration of SC with NT2 cells provides a novel transplantable tissue source, which can be tested to determine if SC will suppress rejection of the grafted NT2 cells and provide for their short- and long-term trophic support in situ in the treatment of experimental PD.
The Italian Space Agency, in line with its scientific strategies and the National Utilization Plan for the International Space Station (ISS), contracted Thales Alenia Space Italia to design and build a spaceflight payload for rodent research on ISS: the Mice Drawer System (MDS). The payload, to be integrated inside the Space Shuttle middeck during transportation and inside the Express Rack in the ISS during experiment execution, was designed to function autonomously for more than 3 months and to involve crew only for maintenance activities. In its first mission, three wild type (Wt) and three transgenic male mice over-expressing pleiotrophin under the control of a bone-specific promoter (PTN-Tg) were housed in the MDS. At the time of launch, animals were 2-months old. MDS reached the ISS on board of Shuttle Discovery Flight 17A/STS-128 on August 28(th), 2009. MDS returned to Earth on November 27(th), 2009 with Shuttle Atlantis Flight ULF3/STS-129 after 91 days, performing the longest permanence of mice in space. Unfortunately, during the MDS mission, one PTN-Tg and two Wt mice died due to health status or payload-related reasons. The remaining mice showed a normal behavior throughout the experiment and appeared in excellent health conditions at landing. During the experiment, the mice health conditions and their water and food consumption were daily checked. Upon landing mice were sacrificed, blood parameters measured and tissues dissected for subsequent analysis. To obtain as much information as possible on microgravity-induced tissue modifications, we organized a Tissue Sharing Program: 20 research groups from 6 countries participated. In order to distinguish between possible effects of the MDS housing conditions and effects due to the near-zero gravity environment, a ground replica of the flight experiment was performed at the University of Genova. Control tissues were collected also from mice maintained on Earth in standard vivarium cages.
Polyethylene terephthalate (PET) was used as the scaffold material to support the proliferation of human mesenchymal stem cells (hMSCs). The cells were cultured either statically in multi-wells or in a spinner flask agitated at 80 rpm for up to 20 days. To optimize the cell expansion condition, effects of the initial cell density and basic fibroblast growth factor (bFGF) were examined. During culture, cell growth and metabolism were tested. After 20 days, cells were harvested and surface markers were identified and quantified with flow cytometry. The results showed that hMSCs seeded at the lowest density gave the highest expansion fold. hMSCs grown in porous three-dimensional (3D) matrices displayed significantly different characteristics in terms of their proliferation and metabolism. PET matrices with 3D space could sustain cell proliferation for a long time. In addition, a low concentration (5 ng mL(-1)) of bFGF significantly enhanced the expansion of hMSCs in PET. Cell attachment and distribution in PET matrices were studied with confocal laser microscopy and scanning electron microscopy, which also confirmed cell proliferation. Furthermore, most of the cells in PET matrices were CD29, CD44 and CD105 positive, and CD34, CD45 and CD14 negative, confirming that hMSCs cultured in 3D PET matrices can be expanded and maintained in their undifferentiated state for at least 20 days without subculturing.
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Decreased bone mineral density (BMD) in astronauts returning from long-duration spaceflight missions has been well documented, but the altered mechanical loading environment experienced by the musculoskeletal system, which may contribute to these changes, has not been well characterized. The current study describes the loading environment of the lower extremity (LE) during typical days on the International Space Station (ISS) compared to similar data for the same individuals living on Earth. Data from in-shoe force measurements are also used as input to the enhanced daily load stimulus (EDLS) model to determine the mechanical "dose" experienced by the musculoskeletal system and to associate this dose with changes in BMD. Four male astronauts on approximately 6-month missions to the ISS participated in this study. In-shoe forces were recorded using capacitance-based insoles during entire typical working days both on Earth and on-orbit. BMD estimates from the hip and spine regions were obtained from dual energy X-ray absorptiometry (DXA) pre- and post-flight. Measurable loading was recorded for only 30% of the time assigned for exercise. In-shoe forces during treadmill walking and running on the ISS were reduced by 25% and 46%, respectively, compared to similar activities on Earth. Mean on-orbit LE loads varied from 0.20 to 1.3 body weight (BW) during resistance exercise and were approximately 0.10 BW during bicycle ergometry. Application of the EDLS model showed a mean decrease of 25% in the daily load experienced by the LE. BMD decreased by 0.71% and 0.83% per month during their missions in the femoral neck and lumbar spine, respectively. Our findings support the conclusion that the measured ISS exercise durations and/or loading were insufficient to provide the loading stimulus required to prevent bone loss. Future trials with EDLS values closer to 100% of Earth values will offer a true test of exercise as a countermeasure to on-orbit bone loss.
Effect of gravity on human spontaneous 10-Hz electroencephalographic oscillations during the arrest reaction
Electroencephalographic oscillations at 10 Hz (alpha and mu rhythms) are the most prominent rhythms observed in awake, relaxed (eye-closed) subjects. These oscillations may be considered as a marker of cortical inactivity or an index of the active inhibition of the sensory information. Different cortical sources may participate in the 10-Hz oscillation and appear to be modulated by the sensory context and functional demands. In microgravity, the marked reduction in multimodal graviceptive inputs to cortical networks participating in the representation of space could be expected to affect the 10-Hz activity. The effect of microgravity on this basic oscillation has heretofore not been studied quantitatively. Because the alpha rhythm has a functional role in the regulation of network properties of the visual areas, we hypothesised that the absence of gravity would affect its strength. Here, we report the results of an experiment conducted over the course of 3 space flights, in which we quantified the power of the 10-Hz activity in relation to the arrest reaction (i.e., in 2 distinct physiological states: eyes open and eyes closed). We observed that the power of the spontaneous 10-Hz oscillation recorded in the eyes-closed state in the parieto-occipital (alpha rhythm) and sensorimotor areas (mu rhythm) increased in the absence of gravity. The suppression coefficient during the arrest reaction and the related spectral perturbations produced by eye-opening/closure state transition also increased in on orbit. These results are discussed in terms of current theories on the source and the importance of the alpha rhythm for cognitive function.