Advantage of Animal Models with Metabolic Flexibility for Space Research Beyond Low Earth Orbit

Abstract

As the world's space agencies and commercial entities continue to expand beyond Low Earth Orbit (LEO), novel approaches to carry out biomedical experiments with animals are required to address the challenge of adaptation to spaceflight and new planetary environments. The extended time and distance of space travel along with reduced involvement of Earth-based mission support increase the cumulative impact of the risks encountered in space. To respond to these challenges, it becomes increasingly important to develop the capability to manage animal safety and well-being during transportation and research experiments in space. One approach may be to take advantage of an organism's self-regulatory system, which enables them to better adapt and mitigate harmful environmental factors encountered in spaceflight. Recent technological advances have enabled researchers to suppress or enhance metabolism “on demand” in a variety of animal species. These animal models can be used as “pathfinders,” which are capable of tolerating the environmental extremes exhibited in spaceflight, including altered gravity, exposure to space radiation, chemically reactive planetary environments, and temperature extremes. In this report, we survey several of the pivotal metabolic flexibility studies and discuss the importance of utilizing animal models with metabolic flexibility with particular attention given to the ability to suppress the organism's metabolism in spaceflight experiments beyond LEO. The presented analysis demonstrates the adjuvant benefits of these factors to minimize damage caused by exposure to spaceflight and extreme planetary environments. Examples of microorganisms and animal models with dormancy capabilities suitable for space research are considered in the context of their survivability under hostile or deadly environments outside of Earth. Potential steps toward implementation of metabolic control technology in spaceflight architecture and its benefits for animal experiments and manned space exploration missions are discussed.

Introduction

While the increased human presence in orbit over the last four decades has shown that humans can adapt to short-duration spaceflight, we still have an incomplete understanding of the adaptation to long-duration spaceflight, and little is known about health-related consequences of long-term exposure to the spaceflight environment. Future missions to the Moon, Mars, and other deep space objects such as asteroids or moons of other planets provide extraordinary scientific opportunities for space biologists to explore life's ability to adapt to the spaceflight environment during long-duration missions. By studying experimental animals aboard deep space missions, scientists can better understand the adaptive response of life to long-duration spaceflight. Results of these missions can help to define the requirements for optimal human health in deep space as well.

Since the of era of orbital flights, animals have always preceded humans in space missions to act as “pathfinders,” to help scientists produce new medical knowledge and test engineering design concepts that are required to support human space exploration. Animal models are also recognized as cost-effective solutions to probe fundamental biology questions related to human health. Before Apollo missions to the moon, animals were used only once in space missions beyond Low Earth Orbit (LEO). In 1968, a pair of Russian tortoises with a number of other biological specimens launched, on a translunar mission aboard the Zond 5 spacecraft, were the first animals from Earth that passed within 1,950 km of the lunar surface and returned safely to Earth.¹ This spacecraft was planned as a precursor to a manned lunar spacecraft. The next time animals were sent beyond LEO was aboard the Apollo 17 mission, launched on December 17, 1972, with the main objective to gain a better understanding of cosmic particle radiation on animal tissues. In this mission, most of the pocket mice chosen for the flight experiments successfully survived the 13-day journey after orbiting the moon.²

As long-term exploration missions by space agencies and commercial entities continue to evolve and expand, we must design novel approaches to carry out biomedical experiments with animals based on the necessity of their long presence in space. The payoff will be a significant improvement in selecting and designing methods for optimizing adaptation to long-duration spaceflight.

A historical review of all animal experiments in spaceflight research since their first use in the 1950s shows that mortality of animals due to unpredictable failures in the life support systems remains the main issue of concern, being higher than the expected 5% level.³ Although mortality risk among animals in space is significantly minimized in the currently available animal habitat hardware systems developed by the National Aeronautics and Space Administration (NASA), Japanese Aerospace Exploration Agency (JAXA), and the European Space Agency (ESA) to support animal research on International Space Station (ISS), mortality rates still remain high, especially in case of nonmanned autonomous missions.^3,4 Since extended-duration spaceflight experiments beyond LEO will likely contribute more complications to animal welfare, it becomes important to design novel approaches to ensure animal safety, scientific return, and mission outcome. One approach may be to take advantage of altering an organism's self-regulatory control system, which could enhance animal survival in extraterrestrial environments.

In this report, we will survey some pivotal studies and discuss the importance of utilizing animal models with metabolic flexibility in spaceflight experiments beyond LEO. The presented analysis demonstrates the adjuvant benefits of this selection factor to minimize damage caused by exposure to spaceflight and extreme planetary environments, including altered gravity, elevated radiation, chemically reactive planetary dusts, and temperature extremes.

The information, concepts, and hypotheses summarized are the basis for a new metabolic control strategy, and its possible incorporation into the future spaceflight architecture beyond LEO. It is also important to note that this strategy is not limited to the category of animals with naturally occurring metabolic flexibility such as hibernating animals. With the current state of biotechnology, many other species, which are not normally capable of inducing metabolic depression, could be intentionally preconditioned to this state using the metabolic control technology. The possibility of manipulating metabolic mechanisms in animals and testing them in long-duration missions beyond LEO may lead to development of biomedical technologies capable of sustaining and protecting astronauts from the extreme dangers of the spaceflight environment. Performing hypometabolic studies on animals would be a precursor to performing similar studies on humans and could help inform researchers about the development of countermeasures designed to protect the crew in future long-duration human space exploration missions.

Metabolic Control as a Strategy for Transportation of Experimental Animals Beyond Leo

With growing interest in spaceflight beyond LEO, it becomes increasingly important to ensure the health and physiological performance of animal models in spaceflight. Upmass and power constraints are of paramount importance in considering the logistics of transporting experimental animals into space. Current animal life support technologies have large-volume and mass requirements. In the context of the emerging space and planetary architecture, these constraints are expected to continue and are a limiting factor for NASA in its efforts to develop a portfolio of deep space biological research that can answer key questions of interest to both the Exploration Systems Mission Directorate and the Science Mission Directorate. One approach to reduce or eliminate expensive life support technologies for nominal animal metabolism during long-duration spaceflight would be to apply the principles of metabolic control on model organisms. By altering the metabolism of animals to a minimal level during a spaceflight mission, life support requirements are reduced until normal metabolism rates are desired. The phenomenon of the metabolic flexibility, or the ability to reversibly alter metabolism in response to availability and need for energy, is well known for many simple and unicellular organisms.^5–8

Metabolic suppression is defined as a drop in standard metabolic rates to less than the normal value with energy-saving benefits toward survival in response to life-threatening environmental stressors. The molecular mechanisms that regulate reversible transitions to and from hypometabolic states are conserved among biologically diverse organisms and include the coordinated reduction of specific cell processes at the genetic, molecular, cellular, organ, and organismal levels.^9–11

Metabolic flexibility in its different forms, extent, and duration is also observed in complex organisms and could be utilized in spaceflight as well. Hibernation, estivation, torpor, diapause and its extreme form cryptobiosis, when an organism shows no visible signs of life, are good examples of behaviors that may be useful for long-duration spaceflight. Changes in metabolic rate of some organisms have been observed to be reduced by 80%, and nearly 100% in cryptobiotic animals.^10,12 Metabolic suppression is also evident in human-sized animals such as the black bear, giant panda, and even to some degree in human.^13–15

Although metabolic suppression is a natural survival response to changes in environmental conditions, it can also be induced in some organisms by altering factors such as temperature or through exposure to selective chemical agents.^6,7,16–19 With recent advances, it is now possible to deliberately initiate and end dormant states in a variety of animal species that do not naturally hibernate, demonstrating the achievement of metabolic control in a variety of “nonhibernating” species.^{6,7,17,20–23}

Carefully controlled studies that precondition organisms to survive hostile environmental conditions create an opportunity for scientists to investigate metabolic control of organisms for specific purposes related to long-duration spaceflight. In the metabolically suppressed state, animals have practically no response to any environmental factors, including pathologies and chemical toxicity, which creates an ability to potentially minimize or even exclude their impacts. This may be particularly useful in long-duration spaceflight, and in extraterrestrial planetary environments of altered gravity, radiation, and dusts. By using animal models that can be metabolically controlled, scientists will be able to compare the practical advantages of hibernating organisms to currently used animal models.

In planning for long-duration space missions, the availability of oxygen, water, and food is critical for survival. Intentionally induced metabolic downregulation may be a useful operational response when the available oxygen and/or food supply are limited. For example, past studies have revealed significant differences in metabolic rate, measured by consumption of oxygen for mice in active and metabolically suppressed states.²⁴ On average, active dormice at 24°C consume 2.08 mL/g/h, while hibernating dormice consume only 0.008 mL/g/h, demonstrating how animals with metabolic flexibility dramatically reduce consumption of vital resources.

Another profound hallmark of metabolic suppression is a quantifiable reduction in food intake during the stasis. Many non-hibernating animals, which have been used in spaceflight experiments including rodents, do not possess extensive metabolic energy reserves and therefore rely on small fuel stores when food is limited or absent. These animals are in serious danger of death if a source of food is not found quickly. Therefore, in the absence of food, intentionally induced metabolic suppression may prolong animal survival over extended periods of time.

Since humans first starting using animals in space experiments in the 1950s, animal mortality has been a problem in some missions. However, much can be learned from those that survived in missions that experienced hardware problems. For example, in the BION M1 mission, the animal mortality rate was observed to be about 75%. It was found that all gerbils, most of the 45 mice, and all of the fish did not survive the mission due to equipment failure. In contrast, all geckos and snails onboard BION-M1 did survive the flight, which demonstrates the tremendous tolerance they have to hostile environmental conditions and limited consumables.⁴ Despite the high mortality rate, the BION-M1 mission has provided the scientific community important information and useful data on the limits of survivability of organisms when exposed to unpredictable problems in life support systems. The mission also highlights the importance of the selection process for the animal models used in flight to reduce risk to animals.

If animal models capable of metabolic control are selected for short- or long-duration space biology experiments, it is possible that likelihood of mission success will be enhanced if equipment problems occur. For example, fasting mice that are completely deprived of food but have access to water, can survive only 2–5 days depending on strain differences,^25,26 while dormice or pocket mice, which belong to the category of animals with metabolic flexibility, can survive without food and water up to 11 months when hibernating.^25,27 Therefore, animals with flexible metabolism may be able to better tolerate unexpected hardware failures that result in life-threatening limitations in consumables and environmental factors.

In addition, if the mass and power requirement of life support systems can be reduced because animal models with flexible metabolism are chosen, transportation of the experimental animals to other planets and deep space destination becomes more feasible.

In space biology, differentiating between the biological effects of launch or reentry versus fundamental responses to microgravity has always been a problem. However, this challenge could be overcome if animals were metabolically preconditioned for both launch and the return phases of a mission. By inducing a metabolically controlled state, biological responsiveness to the environmental factors and stress related to launch and reentry could conceivably be eliminated. In a hypothetical experimental design, animals could be activated to a “normal” physiological state during the desired spaceflight phase allowing researchers to focus completely on spaceflight effects not related to launch or reentry.

Furthermore, payloads that contain highly flexible organisms have significant advantages over payloads with organisms that have limited tolerance and extensive requirements. Placing animals into a reversible hypometabolic stasis before launch will also help to solve many preflight experiment operational problems related to unpredictable launch delays.

The study of metabolic control in spaceflight will help to address questions formulated in the NASA Fundamental Space Biology (SB) Science Plan and the National Research Council's (NRC) 2011 Decadal Survey Report question, “How are the basic metabolic rate and metabolism of living systems, including life span, affected by spaceflight?” Knowledge gained from the use of animals in suspended animation will provide opportunities to gain insight into the development of mitigation strategies designed to reduce risks associated with long-duration human spaceflight. The development of solutions to other biomedical problems where metabolic control is needed may also emerge from this work.

Examples of microorganisms and animal models with dormancy capability suitable for space research are summarized below. Each of these organisms possesses unique metabolic mechanisms and molecular machinery that enable them to survive in what would normally be considered hostile or deadly environments in very unusual ways. These creatures demonstrate features useful for spaceflight metabolic control studies and could provide synthetic biologists information on how to engineer organisms designed to be metabolically controlled during long-duration spaceflight.

Life Forms Capable to Adapt to Adverse Conditions Beyond Earth

Microbes are the most diverse and abundant type of organism on Earth. They possess billions of years of evolutionary adaptions that have enabled them to survive in the extraordinary wide range of physiochemical environmental conditions that exist above, on, and within the Earth. Microbes also have the ability to suppress metabolism and remain dormant for long periods of time, and routinely use this capability to overcome unfavorable environmental conditions. Approximately 99% of all microbes on Earth are in a dormant state,^28–30 and only a tiny fraction is metabolically active at any given time. That dormancy and the presence of such large reservoirs of microbial biodiversity have important implications for the stability and functioning of ecosystem services over both short term and geologic time. Consequently, microorganisms that are capable of switching to and from the dormant state may outcompete other microorganisms.³¹ Microbes may also be involved in panspermia. Microbe-colonized rocks could be ejected from Earth during impact processes and fall onto other planets or Moons. Similarly, because rocks from other planets, the Moon, and asteroids have landed on the Earth due to impact processes, it becomes possible to imagine microbes being transported across the solar system from planet to planet via impact processes. It is reasonable to expect that microbes in a dormant metabolic state may be ideally suited to survive such processes. Evidence of dormancy in microbial species on Earth is a clue that suggests a preference for more favorable environmental conditions that existed in the past. This has implications for panspermia and the search for life on Mars, Europa, Enceladus, or other astrobiological targets.

Over last two decades, a great deal of evidence has amassed that demonstrates primitive life forms can survive in space, and even remain viable following direct exposure to the vacuum of space. NASA, ESA, and the Russian Space Agency have conducted experiments with a variety of organisms that were exposed to the hostile environment of outer space, including naturally occurring space radiation, extraordinary temperature, and pressure variations. Some microbes demonstrated remarkable survivability. Moreover, many of those that did survive were not expected to tolerate these environmental conditions whatsoever.³¹ The organisms tested included spores of Bacillus subtilis,^32–35 the lichen Rhizocarpon geographicum, Xanthoria elegans,³⁶ and adults and eggs of the tardigrades Richtersius coronifer and Milnesium tardigradum.³⁷ B. subtilis (70%) spores survived 2,107 days in space, when shielded against solar ultraviolet radiation.³⁸ Recently, Russian cosmonauts have found traces of sea plankton and microscopic particles on the exterior surface of the ISS.³⁹ If such findings can be confirmed, this will provide additional evidence that some organisms can survive in low-gravity, extreme-temperature conditions and hard cosmic radiation.

Results from other spaceflight experiments and ground studies suggest that terrestrial microbes may be able to both survive a trip to Mars aboard a spacecraft and adapt to a new planetary environment under special circumstances.^40,41

Terrestrial environments outside of the Earth are exposed to large doses of solar and cosmic radiation, are extremely hot, cold, or experience wide temperature swings, are extremely dry, and are hypersaline. For example, hypersaline environments have been observed on Mars and are expected in the oceans that exist beneath the frozen surfaces of Europa and Enceladus, the moons of Jupiter and Saturn.^42–46 Species that have ability to adapt and survive in analogous conditions on Earth include algae, protozoans, fungi cysts, and bacteria. Some demonstrate cryptobiosis and long periods of dormancy in Antarctic and Arctic ice.⁴⁷ Novel ecologies recently discovered in salt crusts of the United Arab Emirates⁴⁸ may also contain microbes suitable for metabolic control studies in hypersaline environments.

It is remarkable that several groups of species among the Tardigrade Ramazzottius and Ozobranchus jantseanus are able to survive extremely low temperatures by reversibly suspending their metabolism and going into a state of cryptobiosis.⁴⁹ Although these species are not considered extremophilic because they are not normally in “extreme” environmental conditions, their ability to survive when exposed to them is scientifically interesting and may be useful for space biology, astrobiology, and exobiology research purposes.

It also should be noted that although the surfaces of many extraterrestrial objects are likely sterile and not habitable, the subsurface and interiors of them may be habitable. Life forms that might exist in the subsurface of extraterrestrial bodies would be protected from harmful conditions on the surface.⁵⁰

Characterization of life forms capable to adapt and reproduce in adverse conditions, such as high- and low-temperature environments, is an important aspect of astrobiology research. Moreover, studies that explore the ability of microbes to survive on materials observed on other worlds such as Mars⁵¹ are providing new insight into the ability of life from Earth to exist on present-day Mars. However, we must consider the possibility that extraterrestrial environments may be so extreme that they prevent all metabolic processes. Nevertheless, it is important to study microbial survival strategies in hot, cold, hypersaline, and desert environments to better understand the limits of life and the potential for it to exist elsewhere in the solar system. If life exists on Mars, it likely exists in a dormant, metabolically controlled state within or underneath rocks, or in the subsurface, in ice, or salt-rich conditions.

Lichens

Lichens are symbiotic associations (holobionts) between fungi (mycobionts) and certain groups of cyanobacteria or unicellular green algae (photobionts). This symbiotic association was essential in the colonization of dry terrestrial habitats.⁵² The vast majority of lichens are desiccation tolerant and can survive in a suspended animation during long periods of drought until water becomes available again, at which time they revive and resume normal metabolism.^52,53 On rehydration they recover normal photosynthetic rates within a very short time span. Individual lichens can live for hundreds or even thousands of years.⁵⁴

Lichens also demonstrate a unique symbiotic survival strategy. The fungus creates a niche for its algae, in places where the algae alone would likely not survive. Occasional exposure to water (rain, flooding) allows them to recharge and store food for the next period of dormancy. Because lichens enable algae to live all over the world in many different climates, they also serve as an important role in the carbon cycle and also produce oxygen. Algae, lichens, and mosses take up approximately 14 billion tons of carbon dioxide and approximately 50 million tons of nitrogen per year from atmosphere and fix it onto the Earth's surface.⁵⁵ Studies exposing lichens to simulated martian conditions were recently performed to characterize lichen metabolic responsiveness.⁵⁶

Snails

Invertebrates such as snails are convenient test subjects for biological research due to their low weight, cost, and wide range of metabolic activity. Snails are able to survive harsh environments, including cold, hypergravity, hypogravity, and high doses of ionizing radiation (up to 200 Gy).⁵⁷ Snails have been flown on several spaceflights (NASA, ESA, Russian, Chinese) for studies of readaptation to Earth's gravity following return from space. The edible snail (Helix pomatia L.) is able to suppress its metabolism in response to harmful environments such as starvation and hibernate for years.⁵⁸ Some snails such as Oxystyla pulchella are able to increase their life span sevenfold by hibernating from 3 to 23 years.⁵⁹ This unique ability gives them significant survival advantages in response to life-threatening conditions when compared to animals with no metabolic flexibility. For example, snails were among the survivors of the 2013 BION-M1 mission that experienced technical problems.⁴

Taken collectively, we have learned that snails are excellent candidates for spaceflight metabolic control experiments. Snails can be placed in a stasis, transported to LEO and beyond, where they could be later revived for physiological experimentation.

Leeches

The O. jantseanus leech found in freshwater turtles in East Asia is able to tolerate extraordinary temperature extremes. In comparison to Tardigrade Ramazzottius, and the larvae of the Chymomyza costata, which can survive for up to an hour in liquid nitrogen, O. jantseanus leeches have survived 2.5 years at a temperature of −90°C and in a liquid nitrogen for a full day. The leech was also capable of enduring repeated freeze–thaw cycles in the temperature range from 20°C to −100°C and then back to 20°C.⁶⁰

Arctic Caterpillars and Ice Worms

Arctic woolly bear moth and ice worms are found in Greenland and Canada around the Arctic Circle. Unlike the other caterpillars, Gynaephora groenlandica caterpillars spend about 5% of their lives eating, feeding on the Arctic tundra during the month of June. For the rest of their lives they are dormant. When exposed to freezing temperatures, the caterpillars break down all their mitochondria, alter their metabolism, and start to synthesize glycerol, which acts as an antifreeze to protect the cells from the freezing conditions. In this adaptive state, Gynaephora is able to survive in temperatures below −60°C. As a result, the woolly bear caterpillar has the longest life cycle of any butterfly or moth. It can take up to 14 years for the insect to complete metamorphosis, from egg to adult moth.⁶¹

The ice worm, Mesenchytraeus solifugus, is among a few metazoan species that not only survives at subzero temperatures but also has the ability to maintain all biological processes at 0°C. Ice worms remain fully functional at freezing temperatures. Compared to their mesophilic counterparts, their response to temperature change is distinctly opposite, namely, ice worms increase production of ATP to offset, at least in part, the inherent lethargy and death usually associated with cold temperature.⁶² Although they both live in similar conditions, the woolly bear caterpillars and the ice worms both use very different strategies to survive the frost. The ice worms alter their internal structure and metabolism to allow them to function at freezing temperatures, rendering them permanently incapable of living anywhere else.

Turtles

The Russian turtle (Agrionemys horsfieldii) has been used in LEO experiments and also been flown around the Moon. Launched on September 15, 1968, the Soviet spacecraft Zond 5 containing two turtles (steppe tortoises), along with other biological payloads, traveled around the Moon and safely returned to earth 6 days later.⁶³ The turtles appeared to tolerate spaceflight well but experienced a 10% weight loss. Tortoises were sent to space again, aboard Soyuz 20, in 1975. That mission kept tortoises in space for 90.5 days, setting a duration record for animals in space.^64,65 In February 2010, the Iranian Space Agency also utilized turtles in its first biological payload into a suborbital flight (Kavoshgar 3).⁶⁶

One of the advantages of turtles as an animal model in space experiments is their ability to combine their low metabolic rate with the ability to further reduce metabolism in response to harsh environmental conditions.⁶⁷ This metabolic depression conserves energy, which lengthens the time turtles can survive in hostile conditions and allows them to emerge from these conditions. Turtles in the Zond 5 mission were able to not only survive the round trip to Moon but also survive a ballistic 20 G during reentry to earth.^1,68

Mouse

Rodents have been the most frequently flown mammalian animal model used to study physiological responses to the space environment. Mice are broadly used to study effects of low gravity and space radiation on the musculoskeletal system and cardiovascular and immune functions.^69–72 Although the deleterious effects of bone loss and muscle atrophy appear to be common to most species in microgravity and also in bed rest experiments on humans, recent studies have shown that animals that undertake extended bouts of natural immobility (i.e., dormancy) consistently demonstrate less atrophy than that experienced by the other mammalian models immobilized for considerably less time.^73–76 Knowledge gained in the use of animals in dormant state in spaceflight could reveal the fundamental mechanisms of adaptation to spaceflight. Such knowledge may provide insights for potential long-duration human spaceflight risk mitigation strategies and potential new approaches for solving space biomedical problems.

Therefore, mice, with intentionally induced metabolic suppression, may be useful subjects for developing experimental models for testing the efficacy of metabolic control and its effect on bone loss and muscle atrophy during spaceflight.^6,7,77 Long-duration studies will allow scientists to explore the possibility of manipulating metabolic mechanisms to reduce the negative effects of multiple space environmental factors on animals and humans during a long-duration space mission.

Dormouse

The dormouse (Glis glis) is a small arboreal rodent that has an extremely long life span of up to 9 years, and is able to survive cold temperatures and food restriction during a hibernation period of up to 8 months. These hibernators have the ability to reduce their metabolic rate to a fraction of their basal energy requirements and lower their body temperature nearly to external ambient temperatures. G. glis genome has recently been sequenced providing researchers opportunities to identify key underlying molecular pathways of its protective metabolic control adaptations.⁷⁸ As a result, the dormouse is an attractive animal model for spaceflight experiments aboard the ISS or free-flying biosatelites.

Pocket Mouse

Perognathus longimembris is another facultative homeotherm with the ability to dramatically suppress its metabolic rate in response to environmental stress.⁷⁹ This species has the ability to produce sufficient quantities of water metabolically from its food and thus does not require a supply of drinking water. Due to this capability as well as its small size, pocket mice were selected for the Apollo 17 moon orbital mission, which demonstrated high rate of survival in the BIOCOR experiments.^80,81 Later, in July 1973, pocket mice were also used as model organisms in the Skylab 3 mission to study the stability of circadian rhythms during orbital spaceflight.⁸¹

Squirrels

Arctic squirrels (Spermophilus parryii) are able to reduce their core body temperature to = 2.9°C in the wild, while keeping their brain temperature just above 0°C.⁸² The physiological changes in the brain of hibernating squirrels are most likely caused by the effects of low temperatures resulting in significant reduction of their neural activity. Arctic squirrels lose up to 60% of their neural synapses during hibernation. However, these connections are later rewired when the animal emerges from hybernation.⁸³ The animal experiences a massive resprouting of neural networks, enabling its learning and memory performance to improve. This suggests that the loss of synapses during hibernation could actually be beneficial for learning about the new features of their environment that may have changed over the winter months.⁸⁴

Although they are classically used in hibernation research, Arctic squirrels could be an appropriate model for space biology, to address questions that investigate cognitive integrity after prolonged exposure to spaceflight conditions. For example, scientists have discovered that the protein RBM3 helps synapses to rebuild in animals awaking from hibernation, which restores normal brain activity. Humans also have the protein, but in some clinical examples it appears not to function, such as in populations of people with Alzheimer's disease. Researchers are hopeful that a drug that mimics or increases the effectiveness of RBM3 could help restore lost brain functions for people with dementia. Interestingly, this protein may also be involved in hypothermia. Researchers have found that metabolic suppression due to hypothermia can protect the brain from some forms of damage, and this phenomenon is being investigated for the development of treatment methods for strokes and other forms of acute brain injury. A similar protective mechanism is now also being studied in neurodegenerative disease.⁸⁵ These and other lines of research may help to mitigate or even solve cognitive problems that astronauts may experience during long-term exposure to spaceflight radiation and microgravity. Understanding changes in neurocognitive and neuropsychological parameters that influence astronauts in flight is an area of important study for the NASA Human Research Program, and is of high relevance with regard to future human missions to Mars.

It has been shown that space radiation impairs the cognitive abilities of rodents, suggesting that astronauts who spend extended time in space may suffer similar consequences.⁸⁶ Other environmental characteristics specific to space missions may affect neurocognitive performance presenting risks to mission success. Understanding the neurobiological processes underlying cognitive impairment caused by spaceflight in animal models with metabolic flexibility may allow researchers to identify and develop optimal behavioral and pharmacological countermeasures for astronauts.

Small Primates

The fat-tailed dwarf lemur (Cheirogaleus medius) is a close genetic relative to humans and is the first primate in whom hibernation was observed. The adult lemur mass is ∼160 g and its maximum life span in captivity is nearly 30 years.⁸⁷ It is able to hibernate for 7 months at relatively high environmental temperatures after storing fat in its tail, which provides the energy source needed during dormancy. During hibernation, the animal's metabolic activity decreases to about 2 percent of its normal active metabolism, and its body temperature drops to match the ambient temperature. As a primate, the fat-tailed dwarf lemur is probably the best animal model that can be used to explore the potential for hibernation in humans.

During hibernation that is accompanied by hypothermia, the mammalian brain does not generate the electrical activity that is observed during conventional sleep. That led researchers to hypothesize that the need for sleep may explain why mammals periodically wake up from hibernation. In contrast to most hibernating animals (which rarely or never enter REM sleep during hibernation), the brain of hibernating dwarf lemurs is unique because it enters into REM sleep for unusually long periods.⁸⁸ This may be due to the fact that unlike many smaller hibernators, dwarf lemurs' body temperatures during hibernation do not drop significantly. As a result, these unique neurological features make the organism an attractive model for exploring sleep and temperature–metabolism relationships. Because of its genetic similarity to humans, the underlying regulatory mechanisms for genes in fat-tailed dwarf lemur are very important, and suggest that investigations that study practical hibernation or torpor in humans may be warranted.

Incorporation of the Metabolic Control Strategy in Animal Spaceflight Architecture

Further Development of Methods to Achieve Hypometabolic Stasis or Other Modes of Dormancy

Development of research programs that define the optimum methods for induction of the dormant state and achieving metabolic control in key experimental animal systems is currently needed. Such an effort will support the development of validated protocols that can be used for transportation of metabolically controlled model organisms in space. In contrast to animals with naturally developed metabolic flexibility discussed in this article, the technology to actually induce a state of hibernation in nonhibernating animals is relatively new and is a fast developing area of research. Several new approaches utilizing natural chemical agents were recently demonstrated to be able to artificially induce and significantly extend the duration of the hypometabolic stasis in nonhibernating animals.^{6,7,19,89–91} These findings may create foundations for application of the metabolic control strategies not only in animals with metabolic flexibility but also in all other kinds including humans.

For control purposes, it is imperative that metabolic control strategies be tested both on the ground and in space, and that all planetary protection requirements are satisfied when Earth organisms are brought to other planetary surfaces.

Integration of the Metabolic Control Strategy with Life Support System

Because one of the most profound hallmarks of metabolic suppression is the dramatic reduction in life's vulnerable consumables such as oxygen, water, and food supply, integration of the metabolic control strategy with the close-loop ecological life support system will be a great advantage for extension of its operation. One such system utilizing the manmade ecological system has been already designed and tested not only on animals but also on humans to provide their survival under reduced consumable resources.⁹² Use of these and other life support technologies adapted for use with metabolically controlled organisms could be one of the promising strategies that would enable management of emergencies with limited resource constraints as well as minimization of consumable resupply from Earth.

Over the next 5–10 years, a new animal research program should be created to develop the required knowledge and hardware needed to successfully complete validation tests that demonstrate metabolic control, on Earth, in LEO, on the Moon, on Mars, and in deep space. The program should align with the NRC Decadal Survey on NASA Life and Physical Sciences, support the NASA SB Science Plan and the NASA Human Research Program, and be executable by either robotic- and/or human-tended missions. The program should be designed to have a flexible path or phased approach, allowing discovery to guide follow-on technology and operational scenario development relevant for future long-duration human space exploration. To date, NASA has already demonstrated some interest in the development of such technology, providing funding to SpaceWorks, Inc., through the NASA Innovative Advanced Concepts (NIAC) research program, toward scenario drafting and assessment of engineering concept for this technology in application to human mission to Mars.⁹³

Animal studies incorporating metabolic control technology would ideally address the following objectives: (1) demonstrate animal presence in the environments of deep space as well as on or near the Moon and Mars; (2) demonstrate protective and useful effects of metabolic suppression against harmful effects of space radiation and altered gravity, and transit- and destination-specific environmental conditions at the subcellular and whole organism level; and (3) provide preliminary data and assessments for robotic precursor missions incorporating metabolic control technology in future objectives for space colonization.

Thorough evaluation of the effects of the space environment on model organisms and mammalian systems, and our ability to perform metabolic control, will require a combination of both in situ evaluation and sample return. Small, light-weight physiological monitoring systems will need to be developed to provide critical real-time information as biological experiments unfold. If possible, select biological specimens should also be returned to Earth to allow for detailed studies such as “omics” analyses that are not currently possible in spaceflight.

The benefit of implementing metabolic suppression into operational scenarios may also enable the establishment of complex ecologies on and within other planetary surfaces. For example, by using dormant, “metabolically suspended” plants and animals, one can envision colonization of the nearest planets.⁹⁴ We predict that hypometabolic suspended ecosystems are more suitable for transport in space for extended periods without special care compared with an active ecosystem. Also, storage of seeds and metabolically suspended animals would provide a reserve in case the active part of an ecosystem is negatively affected by unpredictable space hazards.

At this time, there is great public interest in the efforts of NASA, other space agencies, and private companies to send humans to Mars in the near future. However, this ambitious plan is not yet supported by results from animal models. It is simply unknown if humans (or any other organism) will survive a prolonged voyage to and from Mars. Moreover, a report on the ethics of long-term spaceflight prepared for NASA by a panel of scientists at the Institute of Medicine of the National Academies in April 2014 stated that such travel potentially includes a “wide range of risks that are poorly characterized, uncertain, and perhaps unforeseeable.”⁹⁵ Therefore, it is imperative that the feasibility of a humans to Mars mission be first tested with an animal “pathfinder” mission, analogous to the Planetary Society's Living Interplanetary Flight Experiment (LIFE).^96,97 Collectively, a robust program of metabolic control research and technology innovation will help to prepare for eventual human presence beyond LEO, on the Moon, Mars, and beyond. The program should adhere to all planetary protection requirements and support the planetary protection office on existing and future planetary protection concerns. It is important to note that any future NASA animal or human research on other planetary surfaces will require reassessment/revision of current NASA planetary protection requirements.⁹⁸

Footnotes

Author Disclosure Statement

No competing financial interests exist.

References

Tate

. Cosmic menagerie: a history of animals in space (Infographic). Space.com, June 28, 2014.

NASA Johnson Space Center.. Apollo 17 mission report. JSC-07904, March 1973.

Ferreira

. 2014. Animal astronauts have about a 66 percent chance of survival. https://motherboard.vice.com/en_us/article/animals-astronauts-have-about-a-66-percent-chance-of-survival (Last accessed on June 2, 2017).

Pultarova

. 2013. Crew of bion M1 found dead upon landing. Space Safety Magazine, October 20, 2013.

Roth

, Nystul

. Buying time in suspended animation. Sci Am. 2005; 292:48–55.

Blackstone

, Morrison

, Roth

. H₂S induces a suspended animation-like state in mice. Science. 2005; 308(5721):518.

Galicia

, Palma

, Selch

, Gomez

, Griko

. Metabolic control as a strategy for payload cost reduction and mitigation of negative space environmental factors. Gravit Space Res. 2011; 25:54–6.

Mayer

. 2016. Dormancy. In Encyclopædia Britannica. www.britannica.com/science/dormancy (Last accessed on January 23, 2017).

Biggar

, Storey

. The emerging roles of microRNAs in the molecular responses of metabolic rate depression. J Mol Cell Biol. 2011; 3(3):167–75.

10.

Storey

, Storey

. Tribute to

P.L

. Lutz: putting life on ‘pause’ – molecular regulation of hypometabolism. J Exp Biol. 2007; 210:1700–714.

11.

Carey

, Andrews

, Martin

. Mammalian hibernation: cellular and molecular responses to depressed metabolism and low temperature. Physiol Rev. 2003; 83:1153–81.

12.

Guppy

, Withers

. Metabolic depression in animals: physiological perspectives and biochemical generalizations. Biol Rev Camb Philos Soc. 1999; 74:1–40.

13.

Tøien

, Blake

, Edgar

, Grahn

, Heller

, Barnes

. Hibernation in black bears: independence of metabolic suppression from body temperature. Science. 2011; 331:906–909.

14.

Nie

, Speakman

, Wu

, Zhang

, Hu

, Xia

, Yan

, Hambly

, Wang

, Wei

, Zhang

, Wei

. Exceptionally low daily energy expenditure in the bamboo-eating giant panda. Science. 2015; 6244:171–4.

15.

V.T.B.. 1906. Human hibernation: a practice common with famine-stricken districts of Russia. The New York Times, November 19, p. 8.

16.

Dark

, Miller

, Zucker

. Reduced glucose availability induces torpor in Siberian hamsters. Am J Physiol. 1994; 267:496–501.

17.

Nystul

, Roth

. Carbon monoxide-induced suspended animation protects against hypoxic damage in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 2004; 101:9133–6.

18.

Dirkes

, Gulik

TMV

, Heger

. The physiology of artificial hibernation. J Clin Transl Res. 2015; 2:78–93.

19.

Doyle

, Suchland

, Ciesielski

, Lessov

, Grandy

, Scanlan

, Stenzel-Poore

. Novel thyroxine derivatives, thyronamine and 3-iodothyronamine, induce transient hypothermia and marked neuroprotection against stroke injury. Stroke. 2007; 38:2569–76.

20.

Padillo

, Roth

. Oxygen deprivation causes suspended animation in the zebrafish embryo. Proc Natl Acad Sci U S A. 2001; 98:7331–5.

21.

Frappell

, Lanthier

, Baudinette

, Mortola

. Metabolism and ventilation in acute hypoxia: a comparative analysis in small mammalian species. Am J Physiol Regul Integr Comp Physiol. 1992; 262:1040–6.

22.

Lee

. Is human hibernation possible?. Annu Rev Med. 2008; 59:177–86.

23.

Zhang

, Wang

, Luo

, Ji

, Nemoto

, Chen

. When hypothermia meets hypotension and hyperglycemia: the diverse effects of adenosine 5′-monophosphate on cerebral ischemia in rats. J Cereb Blood Flow Metab. 2009; 29:1022–1034.

24.

Wilz

, Heldmaier

. Comparison of hibernation, estiv ation and daily torpor in the edible dormouse, Glis glis. J Comp Physiol [B]. 2000; 170:511–21.

25.

Williams

, Chambers

, Henderson

, Rashotte

, Overton

. Cardiovascular responses to caloric restriction and thermoneutrality in C57BL/6J mice. Am J Physiol Regul Integr Comp Physiol. 2002; 282:R1459–67.

26.

Jensen

, Kiersgaard

, Sørensen

, Mikkelsen

. Fasting of mice: a review. Lab Anim. 2013; 47(4):225–40.

27.

Hoelzl

, Bieber

, Cornils

, Gerritsmann

, Stalder

, Walzer

, Ruf

. How to spend the summer? Free-living dormice (Glis glis) can hibernate for 11 months in non-reproductive years. J Comp Physiol B. 2015; 185:931–9.

28.

Roszak

, Colwell

. Survival strategies of bacteria in the natural environment. Microbiol Rev. 1987; 51:365e379.

29.

Hoehler

, Jørgensen

. Microbial life under extreme energy limitation. Nat Rev Microbiol. 2013; 11:83–94.

30.

Lennon

, Jones

. Microbial seed banks: the ecological and evolutionary implications of dormancy. Nat Rev. 2011; 9:119–30.

31.

Novikova

, Gusev

, Polikarpov

, Deshevaya

, Levinskikh

, Alekseev

, Okuda

, Sugimoto

, Sychev

, Grigoriev

. Survival of dormant organisms after long-term exposure to the space environment. Acta Astronaut. 2011; 68:1574–80.

32.

Horneck

, Bücker

, Reitz

, Requardt

, Dose

, Martens

, Mennigmann

, Weber

. Microorganisms in the space environment. Science. 1984; 225:226–8.

33.

Horneck

, Rettberg

, Reitz

, Wehner

, Eschweiler

, Strauch

, Panitz

, Starke

, Baumstark-Khan

. Protection of bacterial spores in space, a contribution to the discussion on panspermia. Orig Life Evol Biosph. 2001; 31:527–47.

34.

Horneck

. Responses of Bacillus subtilis spores to space environment: results from experiments in space. Orig Life Evol Biosph. 1993; 23:37–52.

35.

Rettberg

, Eschweiler

, Strauch

, Reitz

, Horneck

, Wanke

, Brack

, Barbier

. Survival of microorganisms in space protected by meteorite material: Results of the experiment EXOBIOLOGIE of the PERSEUS mission. Adv Space Res. 2002; 30:1539–45.

36.

Sancho

, de la Torre

, Horneck

, Ascaso

, de los Ríos

, Pintado

, Wierzchos

, Schuster

. Lichens survive in space: results from the 2005 LICHENS experiment. Astrobiology. 2007; 7(3):443–54.

37.

Jönsson

, Rabbow

, Schill

, Harms-Ringdahl

, Rettberg

. Tardigrades survive exposure to space in low Earth orbit. Curr Biol. 2008; 18(17):729–31.

38.

Horneck

, Bücker

, Reitz

. Long-term survival of bacterial spores in space. Adv Space Res. 1994; 14:41–5.

39.

Knapton

. Sea plankton found living outside International Space Station. www.telegraph.co.uk/news/science/space/11049504/Sea-plankton-found-living-outside-International-Space-Station.html (Last accessed on June 2, 2017).

40.

De la Torre

, Sancho

, Horneck

, de los Ríos

, Wierzchos

, Olsson-Francis

, Cockell

, Rettberg

, Berger

, de Vera

J-P

, Ott

, Frías

, González Melendi

, Lucas

, Reina

, Pintado

, Demets

. Survival of lichens and bacteria exposed to outer space conditions – results of the Lithopanspermia experiments. ICARUS. 2010; 208:735–48.

41.

Sugimoto

, Ishii

, Mori

, Shagimardanova

, Gusev

, Kihara

, Hoki

, Sychev

, Levinskikh

, Novikova

, Grigoriev

. Viability of barley seeds after long-term exposure to outer side of international space station. Adv Space Res. 2011; 48:1155–60.

42.

Shapiro

, Schulze-Makuch

. The search for alien life in our solar system: strategies and priorities. Astrobiology. 2009; 9:335–43.

43.

Hegde

, Kaltenegger

. Colors of extreme exo-Earth environments. Astrobiology. 2013; 13:47–56.

44.

Kounaves

. Life on Mars may be hidden like Earth's extremophiles. Nature. 2007; 449:281.

45.

Andrews-Hanna

, Phillips

, Zuber

. Meridiani Planum and the global hydrology of Mars. Nature. 2007; 446:163–6.

46.

Landis

. Martian water: are there extant halobacteria on Mars?. Astrobiology. 2001; 1:161–4.

47.

Goordial

, et al. Nearing the cold-arid limits of microbial life in permafrost of an upper dry valley, Antarctica. Int Soc Microb Ecol. 2016; 10:1613–1624.

48.

McKay

, Rask

, et al. An unusual inverted saline microbial mat community in an Interdune Sabkha in the Rub' al Khali (the Empty Quarter), United Arab Emirates. PLoS One. 2016; 11(3):e0150342.

49.

Becquerel

. La suspension de la vie au dessous de 1/20 K absolu par demagnetization adiabatique de l'alun de fer dans le vide les plus eléve [in French]. C R Hebd Séances Acad Sci Paris. 1950; 231:261–3.

50.

McMahon

, O'Malley-James

, Parnell

. Circumstellar habitable zones for deep terrestrial biospheres. Planet Space Sci. 2013; 85:312–8.

51.

Bywaters

. Perchlorate reducing bacteria: evaluating the potential for growth utilizing nutrient sources identified on Mars, 2016. Proceedings of the 47th Lunar and Planetary Science Conference, abstract 2946, 2016.

52.

Gasulla

, Herrero

, Esteban-Carrasco

, Ros-Barceló

, Barreno

, Miguel Zapata

, Guéra

. Photosynthesis in lichen: light reactions and protective mechanisms, advances in photosynthesis – fundamental aspects. Najafpour

, ed. InTech, 2012. https://cdn.intechopen.com/pdfs-wm/28373.pdf (Last accessed on June 2, 2017).

53.

Kappen

, Valladares

. Opportunistic growth and desiccation tolerance: the ecological success of poikilohydrous autotrophs. In Functional Plant Ecology. Pugnaire

, Valladares

, eds. pp.. New York: Taylor & Francis, 2007, pp. 7–65.

54.

Hale

. Growth. In The Lichens. Ahmadjian

, Hale

, ed. New York: Academic Press, 1973, pp. 473–92.

55.

Elbert

, Weber

, Burrows

, Steinkamp

, Büdel

, Andreae

, et al. Contribution of cryptogamic covers to the global cycles of carbon and nitrogen. Nat Geosci. 2012; 5:459–462.

56.

de Vera

, Schulze-Makuch

, Khan

, Lorek

, Koncz

, Möhlmann

, Spohn

. Adaptation of an Antarctic lichen to Martian niche conditions can occur within 34 days. Plane Space Sci. 2014; 98:182–90.

57.

Rayburn

. The foundations of laboratory safety: a guide for the biomedical laboratory. New York: Springer-Verlag, 1190, p. 189.

58.

Nowakowska

. Hypometabolism in land snails: controlled or passive phenomenon? In Hypometabolism: Strategies of Survival in Vertebrates and Invertebrates. Nowakowska

, Caputa

, eds. Kerala, India: Research Signpost, 2011, pp. 1–17.

59.

Baker

. Jamaican land snails. Nautilus. 1934; 48:6–14.

60.

Suzuki

, Miyamoto

, Kikawada

, Watanabe

, Suzuki

. A leech capable of surviving exposure to extremely low temperatures. PLoS One. 2014; 9(1):e86807.

61.

Kukal

, Duman

, Serianni

. Cold-induced mitochondrial degradation and cryoprotectant synthesis in freeze-tolerant arctic caterpillars. J Comp Physiol B. 1989; 158:661–71.

62.

Napolitano

. Nagele RG, Shain DH. The ice worm, Mesenchytraeus solifugus, elevates adenylate levels at low physiological temperature. Comp Biochem Physiol A Mol Integr Physiol., 2004; 137:227–35.

63.

Antipov

, Gazenko

, Parfenov

, Saksonov

. Results of a biological experiment conducted aboard the automatic station “Zond-5.”. Aerosp Med., 1969; 40:1244–7.

64.

Clement

, Slenzka

. Animals and plants in space. In Fundamentals of Space Biology: Research on Cells, Animals, and Plants in Space. Clement

, Slenzka

, eds. New York: Springer, 2006, Chapter 2, pp. 51–79.

65.

Baikonur LC1.. Encyclopedia astronautica. www.astronautix.com/b/baikonurlc1.html (Last accessed on 4 March 2009 ).

66.

McDowell

. Kavoshgar. Jonathan's Space Page, September 9, 2012.

67.

Jackson

. Hibernating without oxygen: physiological adaptations of the painted turtle. J Physiol. 2002; 543:731–737.

68.

Gray

. A Brief History of Animals in Space. NASA HQ Printing & Design, 1998. https://history.nasa.gov/printFriendly/animals.html (Last accessed on June 2, 2017).

69.

Masini

, Albi

, Barmo

, Bonfiglio

, Bruni

, Canesi

, et al. The impact of long-term exposure to space environment on adult mammalian organisms: a study on mouse thyroid and testis. PLoS One. 2012; 7(4):e35418.

70.

Zhong

, Garman

, Squire

, Donahue

, Rubin

, Hadjiargyrou

, Judex

. Gene expression patterns in bone after 4 days of hind-limb unloading in two inbred strains of mice. Aviat Space Environ Med. 2005; 76(6):530–5.

71.

Lescale

, Schenten

, Djeghloul

, Bennabi

, Gaignier

, Vandamme

, Strazielle

, Kuzniak

, Petite

, Dosquet

, Frippiat

, Goodhardt

. Hind limb unloading, a model of spaceflight conditions, leads to decreased B lymphopoiesis similar to aging. FASEB J. 2015; 29:455–63.

72.

Milstead

, Simske

, Bateman

. Spaceflight and hindlimb suspension disuse models in mice. Biomed Sci Instrum. 2004; 40:105–110.

73.

Fedorov

, Goropashnaya

, Tøien Ø, Stewart

, Chang

, Wang

, Yan

, Showe

, Donahue

, Barnes

. Preservation of bone mass and structure in hibernating black bears (Ursus americanus) through elevated expression of anabolic genes. Funct Integr Genomics. 2012; 12:357–65.

74.

Tinker

, Harlow

, Beck

. Protein use and muscle-fiber changes in free-ranging, hibernating black bears. Physiol Zool. 1998; 71:414–24.

75.

Hershey

, Robbins

, Nelson

, Lin

. Minimal seasonal alterations in the skeletal muscle of captive brown bears. Physiol Biochem Zool. 2008; 81:138–47.

76.

Ivakine

, Cohn

. Maintaining skeletal muscle mass: lessons learned from hibernation. Exp Physiol. 2014; 99:632–7.

77.

Musacchia

, Volkert

, Barr

. Radioresistance in hamsters during hypothermic depressed metabolism induced with helium and low temperatures. Radiat Res. 1971; 46:353–61.

78.

Gazizova

, Tyapkina

, Logacheva

, Nurullin

, Vikhlyantsev

, Ishihara

, Ishioka

, Gusev

. Long-living hibernators: the first glance on the genome's structure and activity in edible dormice. 52th CRYO 2015 Annual Meeting of the Society for Cryobiology, Kazan Federal University, Russia, 2015. http://cryo2015.com/data/program_FNO_A5_Pass15_final_kor8.pdf (Last accessed on October 23, 2016).

79.

Bartholomew

, Cade

. Temperature regulation, hibernation, and aestivation in the little pocket mouse, Perognathus longimembris. J Mammal. 1957; 38:60–72.

80.

Haymaker

, Look

, Benton

, Simmonds

. Biomedical results of Apollo. Chapter 4: The Apollo 17 Pocket Mouse Experiment. NASA SP-368, 1975.

81.

Souza

, Hogan

, Ballard

, eds. Life into space: space life sciences experiments. NASA Ames Research Center 1965–1990. Washington D.C.: National Aeronautics and Space Administration, 1995. NASA Reference Publication-1372 (online version).

82.

Barnes

. Freeze avoidance in a mammal: body temperatures below 0 degree C in an Arctic hibernator. Science. 1989; 244(4912):1593–5.

83.

Von der Ohe

, Garner

, Darian-Smith

, Heller

. Synaptic protein dynamics in hibernation. J Neurosci. 2007; 27(1):84–92.

84.

Weltzin

, Zhao

, Drew

, Bucci

. Arousal from hibernation alters contextual learning and memory. Behav Brain Res. 2006; 167(15):128–33.

85.

Ginsburg

, Belayev

. Biological and molecular mechanisms of hypothermic neuroprotection. In Therapeutic Hypothermia. Mayer

, Sessler

, eds. Boca Raton, FL: Taylor & Francis Group, 2005, pp. 85–141.

86.

Parihar

, Allen

, Tran

, Macaraeg

, Chu

, Kwok

, Chmielewski

, Craver

, Baulch

, Acharya

, Cucinotta

, Limoli

. What happens to your brain on the way to Mars. Sci Adv. 2015; 1(4):e1400256.

87.

Blanco

, Zehr

. Striking longevity in a hibernating lemur. J Zool. 2015; 296:177–188.

88.

Krystal

, Schopler

, Kobbe

, Williams

, Rakatondrainibe

, Yoder

, Klopfer

. The relationship of sleep with temperature and metabolic rate in a hibernating primate. PLoS One. 2013; 8(9):e69914.

89.

Tupone

, Madden

, Morrison

. Central activation of the A1 adenosine receptor (A1AR) induces a hypothermic, torpor-like state in the rat. J Neurosci. 2013; 33:14512–25.

90.

Horton

, Kaftani

, Bruce

, Bailey

, Krober

, Jones

, Turker

, Khattar

, Su

, Bolling

, Oeltgen

. Isolation and partial characterization of an opioid-like 88 kDa hibernation-related protein. Comp Biochem Physiol B Biochem Mol Biol. 1998; 119:787–805.

91.

Scanlan

, Suchland

, Hart

, Chiellini

, Huang

, Kruzich

, Frascarelli

, Crossley

, Bunzow

, Ronca-Testoni

, Lin

, Hatton

, Zucchi

, Grandy

. 3-Iodothyronamine is an endogenous and rapid-acting derivative of thyroid hormone. Nat Med. 2004; 10:638–42.

92.

Gitelson

, Lisovsky

, Macelroy

. Manmade Closed Ecological Systems: A New Means to Solve some Critical Problems of Human Life Support on Earth and in Space. London: Taylor & Francis, 2003.

93.

Bradford

. Torpor inducing transfer habitat for human stasis to Mars. 2013. NASA funded studies in https://www.nasa.gov/content/torpor-inducing-transfer-habitat-for-human-stasis-to-mars (Last accessed on June 2, 2017).

94.

Alekseev

, Sychev

. Effect of Space station conditions on resting egg survivorship and parameters of life cycle in D. Magna. Abstract of COSPAR 2006, Beijing, China, 2006.

95.

Committee on Ethics Principles and Guidelines for Health Standards for Long Duration and Exploration Spaceflights; Board on Health Sciences Policy; Institute of Medicine; Kahn

, Liverman

, McCoy

, eds. Washington, DC: National Academies Press, June 23, 2014. (Last accessed on June 2, 2017).

96.

Warm-flash

, Ciftcioglu

, Fox

, McKay

, Friedman

, Betts

, Kirschvink

. Living interplanetary flight experiment (LIFE): an experiment on the survivability of microorganisms during interplanetary transfer. Workshop on the Exploration of Phobos and Deimos, 2007. www.lpi.usra.edu/meetings/phobosdeimos2007/pdf/7043.pdf (Last accessed on June 2, 2017).

97.

Zak

. The mission scenario of the Phobos-Grunt project. 2011. www.russianspaceweb.com/phobos_grunt_scenario.html (Last accessed on June 2, 2017).

98.

Workshop on planetary protection knowledge gaps for human extraterrestrial missions on March 24–26, 2015. Moffett Field, CA: NASA's Ames Research Center. www.nasa.gov/ames/ppw2015workshop (Last accessed on June 2, 2017).