Animal Welfare Information Center Newsletter, Winter 1995/1996, Vol. 6 No. 2-4 *************************

Laboratory Animals in Space

Life Sciences Research

by
Gary L. Borkowski, D.V.M., M.S.,
Pennsylvania State University, University Park, Pennsylvania

William W. Wilfinger, Ph.D.,
Biotech Express, St. Bernard, Ohio

Philip K. Lane, M.D.,
Lockheed Martin Engineering and Sciences Services Company,
National Aeronautics and Space Administration (NASA) Ames Research Center,
Moffett Field, California

Abstract

Animals have been invaluable in space life sciences research and have contributed greatly to the current database of knowledge in this field. This article presents an overview of the historical involvement of animals, describes the hardware and logistics of flying animal payloads on the space shuttle orbiters, and discusses future plans for animal experiments in space.

Introduction

Since the beginning of modern space exploration, animals have accompanied and sometimes preceded humans as space travelers. Extensive animal experimentation was used in both the United States and Soviet/Russian space programs to collect the medical knowledge and develop and test the engineering design concepts that would be required to support human space exploration. Initially, animals were used as surrogates to test the suitability of the space environment for human habitation. Once it was determined that complex biological organisms could live in space, humans ventured into space, and took animals along as experimental subjects. This situation continues today aboard the space shuttle orbiters, as well as on Russian spacecraft (4,12).

[*ICON*] Figure 1: Physiological Changes Due to Exposure to Microgravity.

Space exploration has advanced significantly over the last five decades and animals continue to be used in microgravity1 investigations. Bjurstedt has recently reviewed some of the adaptive cardiovascular, musculoskeletal, and neurovestibular changes that have been attributed to microgravity exposure (fig. 1) (6). A primary focus of ongoing animal investigations is to determine how gravitational inputs modulate the complex regulatory mechanisms that may be involved in Earth-based diseases such as anemia, osteoporosis, muscular atrophy, and immune system dysfunction (11,15,16). Many of these experiments use rodent payloads that are transported into space aboard the space shuttle. In this report we will briefly survey some of the pivotal animal studies that made human space flight possible and then focus on the flight hardware that is currently used for microgravity animal investigations aboard the space shuttle.

Laboratory Animals Demonstrate That Living Organisms Can Survive in Space

The preliminary physiological and biological testing for aerospace research occurred at the Physiological Research Laboratory at Wright Field in Dayton, Ohio. From 1935 to 1948, Dr. Harry G. Armstrong used animals and humans in ground-based altitude and acceleration experiments. Based on these pioneering studies, the first sub-orbital rocket-powered animal flight occurred in June of 1948 when an anesthetized rhesus macaque (Macaca mulatta) named Albert was launched aboard a V2 rocket at White Sands, New Mexico. There were three additional V2 rocket flights in 1949 and 1950 involving rhesus and cynomolgus (Macaca nemestrina), but none of the animal payloads were recovered alive because of mechanical failures (2,8).

In 1951 and 1952, three Aerobee rocket flights took place, with mice and nonhuman primates as test subjects. The animals on the third flight flew to an altitude of 64.5 km at a speed of 3,200 kph and were exposed to microgravity for 2 minutes. They were successfully recovered and were appropriately deemed "the first living creatures to survive the test program (2,7,8)."

The next significant involvement of laboratory animals in aerospace research occurred during the "space race" of the late 1950's. Sputnik II, a Soviet (bio)satellite launched in November of 1957, carried a dog (Canis familiaris) named Laika, and Sputnik III, IV, and V carried mice, rats and dogs. In the United States in 1958, three separate mouse payloads were flown in the nose cone of Thor-Able rockets. Physiological telemetry data were obtained from the animals during their 20-minute exposure to microgravity. The Bioflight series of 1958-59 contained a squirrel monkey (Saimiri sciureus) named Old Reliable (Bioflight 1), and a rhesus monkey (Abel) and a squirrel monkey (Baker) on Bioflight 2. The Bioflight 1 experiment collected telemetry data on physiological parameters, and the Bioflight 2 payload was successfully recovered. Later in 1959 and 1960, two rhesus monkeys named Sam and Miss Sam were separately launched to an altitude of 84 km and performance data were collected as the animals were exposed to microgravity. The equipment that would be used on the manned Mercury flights was successfully tested on these missions. In 1961, before Alan Shepard's historic ballistic space flight (May 5, 1961), a chimpanzee (Pan troglodytes) named Ham was launched into space in a Mercury capsule that achieved an altitude of 250 km and a range of 662 km. He was monitored with telemetry equipment and performed discrete and continuous avoidance tasks during the flight. Another chimp (Enos) also spent 3 hours in a microgravity environment before John Glenn's orbital flight (February 20, 1962). Once these critical flights were successfully completed and recovered, there was confidence that humans could live and work in space (2,7,17).

During the Apollo era (1960-72), most of the missions did not include animal payloads, as it had already been shown that animals could survive in space. The last lunar mission (Apollo 17) did, however, include the BIOCORE Pocket Mouse Radiation Experiments to study exposure to cosmic particle radiation hazards. Five pocket mice (Perognathus longimembris) were housed in self-sustaining, hermetically sealed, cylindrical aluminum canisters. Richard Simmonds, D.V.M., was instrumental in coordinating this experiment and was involved with the postflight analysis as well. NASA also launched three Biosatellites during the Apollo years, and Biosatellite #3 carried a rhesus monkey (Bonnie) in orbit for 8 days (2,5).

Skylab, the first U.S. orbiting space station, was launched in May 1973 and orbited the Earth until July 1979, when it re-entered the Earth's atmosphere and crashed in western Australia. The Skylab-3 mission, launched on July 28, 1973, used six pocket mice to study circadian rhythms during spaceflight. These mice were housed in individual circular cages and instrumented for telemetry data collection, but a power failure 30 hours into the mission resulted in loss of the experiment (2,5).

This brief historic overview only summarizes the seminal flight experiments in which animals have been used to significantly advance our understanding of gravitational physiology. A detailed and comprehensive chronological review of numerous experiments that have contributed to our current understanding of aeronautical and aerospace medicine can be obtained in other reports (2,5,7,8). It is also important to note that microgravity investigations involving plant and animal tissues have contributed significantly to our current understanding of gravitational biology (9).

Space Shuttle Provides a More Suitable Environment for Animal Research

In 1981, NASA began using the Space Transportation System (STS) to carry payloads and astronauts into space. The space shuttle orbiter is the flight vehicle for this system, that during launch also includes an external fuel tank and a pair of solid-rocket boosters (SRBs). Other components of the STS include the ground facilities where the shuttle is prepared for flight and tracked and monitored during each mission. There are currently four space shuttle orbiters in operation. On April 12, 1981, Columbia was the first orbiter to be launched from Kennedy Space Center. Challenger, Discovery, and Atlantis were subsequently added to the fleet between 1983 and 1985. Endeavour, the newest orbiter, replaced Challenger, which exploded shortly after launch on January 28, 1986 (1,2,18). The diversity of animal payloads that have flown aboard the space shuttle is summarized in figure 2.

All of the animal experiments that have flown aboard the space shuttle have either been housed in the middeck area, or within a laboratory research module specifically configured for the cargo bay. The orbiter middeck is the housing option most frequently used for rodent experiments (fig. 2). The middeck contains 42 lockers for experiments and payloads. When rodent experiments are scheduled for launch aboard the shuttle, one to three lockers are configured with animal enclosure modules (AEMs) (figs. 3 - 4). The AEM was originally developed by General Dynamics Company for the Student Shuttle Flight Program and is managed by the NASA Ames Research Center (ARC) in Moffett Field, California. The AEMs are currently being tested and modified to support future microgravity investigations with mice. The AEM is a small, portable, self-contained, animal holding facility that is designed to fit within a single middeck locker. It can be integrated into the middeck 12-18 hours before launch and recovered within 3-6 hours after the orbiter lands, thereby providing great versatility for the investigator. Each AEM contains sufficient food (rodent food bars) for the duration of the mission as well as an onboard water supply that can be periodically replenished on orbit. Approximately 18 hours before launch, the animals are transferred to an AEM, transported to the launch pad and loaded into a middeck locker. Five to eight rats are normally housed in each AEM, but the absolute number depends on the strain and weight of the animal, as well as the duration of the mission. Longer duration missions require larger food reserves and smaller animal payloads to meet the middeck locker safety weight constraints (5,7).

[*ICON*] Figure 3: NASA Ames Research Center Animal Enclosure Module Components
[*ICON*] Figure 4: Animal Enclosure Module

The AEM can be thought of as a miniature laboratory animal facility in the sense that it contains all of the components that are required for maintenance of the animals during a mission. Daily health checks can be accommodated during flights by opening the locker cover and pulling the AEM from its stowage position within the locker. A transparent plastic cover on the surface of the animal chamber enables the astronauts to observe the animals at any time during the mission. Food and water consumption can be monitored, and the water reservoir bags can be refilled during the flight as required. Although the animals can be easily visualized, the AEMs are tightly sealed, and the animals are not accessible for manipulation or treatment (3,7).The animal cage portion of the AEM consists of a removable rectangular stainless steel mesh screen (24 X 36 X 22 cm). A portion of this cage volume is occupied by a waterbox that can hold up to 1.5 liters of water to supply the AEM lixits. Bonting et al., have recently compared the AEM to the environment recommended in the 1985 NIH Guide to the Care and Use of Laboratory Animals (7). The AEM meets most of the NIH guidelines, except for a somewhat increased housing density and an increased ambient temperature. In launchpad orientation, available floor space is about 710 cm2, with about 14,750 cm3 of habitable space on orbit. Temperatures within the AEM routinely average about 30o C and run 3-5o C warmer than ambient middeck temperatures in the orbiter. The AEM's do not have active thermal control, therefore the temperature within the habitat depends totally on the middeck cabin temperature. A battery-powered internal temperature recorder is used to log the temperatures within the AEM so that a detailed temperature record can be reconstructed postflight. There are four internal lamps, two of which are used during the day period of the 12-hour light:12-hour dark cycle, and two backup lights. The lamps provide an illumination of approximately 14 lux at the center of the animal cage. The lighting timer has a battery-powered clock that is independent of the orbiter power supply to ensure consistent light cycles. Air circulation is accomplished by four fans that pull cabin air to the back of the cage and through a high efficiency particulate air (HEPA)/charcoal filter and into the animal quarters. After the air passes through the cage, it traverses a second filter where all particulate matter and odors are removed before the air is returned to the orbiter cabin. A continuous airflow of about 15-20 cubic feet per minute is achieved with this system. A 28-volt DC orbiter power supply is used to power the various electrical components within the AEM after integration into the middeck locker. During transit to and from the orbiter, the AEM is connected to an external battery pack (7,13).

[*ICON*] Figure 5: Research Animal Holding Facility in Spacelab Module
[*ICON*] Figure 6: Rodent Housing Unit for Spacelab
[*ICON*] Figure 7: Squirrel Monkey Housing Unit for Spacelab

The Spacelab module was developed and built by the European Space Agency (ESA) and is mounted in the orbiter cargo bay when it is flown. This unique international laboratory facility converts into an on-orbit research center that can provide additional animal space for rodents and nonhuman primates. The Research Animal Holding Facility (RAHF), when placed into a standard Spacelab double rack, provides housing space for up to 24 rats (350 g) or four 1-kg squirrel monkeys (figs. 5, 6, 7). The RAHF provides environmental control, food, water, illumination, and waste management control for the animals. In contrast to the animals housed within the AEM, the animal cages can be removed from the RAHF and transported to the General Purpose Work Station (GPWS). The GPWS is a laminar flow workbench that has glove ports for two astronauts to simultaneously work in the unit. Inside the GPWS the animal cages can be opened and the animals can be removed for tissue or fluid sample collection, the administration of specific treatments, or euthanasia and tissue collection (3,5,7).

The animals are transported and loaded into the RAHF 36 hours prior to launch. To accomplish transfer of the animals from the middeck entry portal to the Spacelab, a Module Vertical Access Kit (MVAK) is used. The MVAK uses a system of ropes and pulleys to lower the technicians from the middeck entrance portal through the orbiter airlock and tunnel adapter and into the Spacelab module while the orbiter is in the vertical position on the launchpad. The cage assemblies containing the experimental animals are then transferred into the Spacelab module and loaded into the RAHF. Individual RAHF rodent cage assemblies are designed to house two rats. Each cage provides a habitat space of 10.8 X 10.8 X 26 cm, uses rodent food bars as a nutrient source, and contains two water lixits. The RAHF water supply, food cassettes and detachable rodent waste management tray assemblies can all be changed out and replenished on orbit (2,3).

The primate housing units are also designed to interface with the RAHF control module. A door on the front of the cages permits limited access to the animals. Each cage is equipped with an emergency restraint mechanism that enables the astronauts to restrain the animals in-flight. Because of the limited number of primates that can be accommodated within the RAHF and the resulting effect this factor has on experimental designs, the primate cage modules have to date been used only for the Spacelab-3 mission (STS-51B, April 1985), and there are no current plans to use primates again on any projected shuttle mission through the turn of the century (2).

The AEM and RAHF are the only flight-certified hardware that can be currently used for warm-blooded vertebrate animal experimentation aboard the orbiter. Due to the size and unique requirements of the hardware, all animal experiments are flown either in the middeck area of the orbiter (AEM) or in the Spacelab module (RAHF). Upgrades to the AEM under consideration include on-orbit food replenishment capability and connection of temperature monitors within the AEM cage to the orbiter data system, to permit realtime downlink2 of in-cage temperatures. The Flight Payloads Office of the Life Sciences Division at NASA Ames Research Center (ARC) is currently designing the Advanced Animal Habitat (AAH), which is scheduled to replace the AEM in 1998. The AAH environment will have active heat rejection and will maintain temperatures in the 22o C-28o C range. Other capabilities in the AAH include on-orbit food and water replenishment, on-orbit animal access, built-in video monitoring capability, and realtime data downlink capability.

The SLSPO (Space Life Sciences Payload Office) foodbar diet was developed over the last 15 years at ARC in support of rodent spaceflight experimentation. It is composed of a dry rodent diet (NASA Experimental Rodent Diet #93062) prepared by Harlan Teklad (Madison, WI), supplemented with minerals and vitamins, and then formed and extruded into bars with a final water content of about 26 percent. The foodbars are then vacuum-sealed in plastic and radiation-sterilized. The foodbars have been successfully used with rats and are currently being evaluated for use with mice.

Ground Control Flight Simulation And Animal Monitoring

The location of the animals aboard the orbiter determines the type and degree of monitoring and interaction that can occur between the mission specialists and the animals. Animals that are housed in the middeck lockers are not accessible to the shuttle crew because the AEMs are securely sealed after the animals are loaded into the cages. Daily observations by the mission specialists are limited to opening the locker door, sliding out the AEM, and observing the animals through the transparent cover. These observations are recorded in log books and also downlinked to the payload scientists for evaluation. Animal health and activity, food and water supplies are monitored during the flight, and the water reservoir is refilled as necessary.

Since October 1992, middeck temperature, humidity, and gas pressure data have been downlinked to the Life Sciences Support Facilities (LSSF) at Kennedy Space Center (KSC). The data are collected and used to control the Orbital Environmental Simulator (OES) where the ground control AEMs are housed. Because of the time delays associated with the downlink of orbiter data, the ground control animals are processed and handled in a manner identical to the flight animals on a 24- or 48-hour time-delay basis. Using the downlink data, the OES is automatically controlled by a system of computers to emulate the middeck environment (temperature, CO2 fraction, relative humidity) aboard the orbiter. The OES is not capable of mimicking the pressure and gas composition changes that occur if there is extravehicular activity (EVA) during the mission.

The ability to manipulate animals housed in cages in the RAHF is a significant advantage for the experimenter. However, until the Space Life Sciences-2 (SLS-2) mission (STS-58, October 1993), this important experimental intervention had not been exploited. Since the RAHF hardware provides more sensitive environmental control and monitoring capabilities, ground control studies can be performed with greater fidelity than is currently possible with the AEM.

Although current procedures with the ground control animals can simulate most orbiter low earth orbit environmental parameters except microgravity and radiation exposure, there are currently no facilities at KSC to mimic the noise (up to 120dB) and the g-forces of launch (3g) and landing (2g). There have been no reported or observed detrimental health effects in the animals as a result of exposure to these stressors, but this dissimilarity between the ground controls and the flight animals must be considered for experimental planning.

The primary landing site for the orbiters is the landing strip at KSC, with NASA's Dryden Flight Research Center at Edwards Air Force Base (near Mojave, California) serving as the alternate site (1). If landing occurs at KSC, animals are removed from the orbiter within 3-6 hours, transported to the LSSF, examined, and handled according to experimental protocols. If bad weather at KSC or technical problems force a Dryden landing, a backup scientific team receives and examines the animals. At that time, several options are available: (1) fly the primary science team from Florida to California to perform postflight procedures, (2) have a full science team in California perform postflight procedures on the flight animals, while the KSC science team performs postflight procedures on control animals in Florida, (3) fly the flight animals from California to Florida for postflight analysis in Florida, (4) fly the control animals from Florida to California for postflight analysis in California, or (5) fly the flight animals from California and the ground control animals from Florida to the principal investigator's laboratory.

International Space Station Alpha

The current plans for International Space Station Alpha include several options for short- and long-term microgravity housing of experimental animals. Currently, there are plans to include a 2-meter diameter variable-g centrifuge facility (maximum 1g) aboard Space Station Alpha to allow for in-flight control animals. The centrifuge rodent housing hardware is currently planned as an upgrade to the AAH, which will provide caging for up to twelve 200-gram rats in a gang-housed environment. On-orbit access to animals, active temperature control, video monitoring, and food and water replenishment will be incorporated in such hardware. Housing hardware for other species, and laboratory facilities for on-orbit collection and analysis of specimens, will be incorporated in the Space Station Life Sciences Suite. The Space Station, once operational, will significantly improve the capabilities to perform animal-based experiments in a microgravity environment.

Conclusions

The ability to conduct life sciences experimentation in space has been pivotal to our understanding of how biological processes are affected by microgravity. The early animal space explorers paved the way for humans to venture into space. A variety of animal models have been used to evaluate an assortment of flight issues that have included propellant systems, radiation exposure, life support systems, and recovery procedures. In the absence of animal models, this work would have progressed much more slowly and with far greater human risk.

Currently, animals often accompany astronauts on space shuttle flights, and they are being used to further our understanding of biological changes that occur during microgravity exposure (10,14,20). It is now known that weightlessness produces certain physiological changes that may produce useful experimental models for studies of Earth-based diseases such as osteoporosis, immune dysfunction, vestibular disorders, wound healing impairment, anemia, and aging (19). The judicious use and application of experimental animal models to the study of complex biomedical and pathophysiological problems will continue to provide new insights into biological mechanisms that influence our lives on Earth and in space3.

Endnotes

1) Microgravity - A term commonly applied to a condition of free-fall within a gravitational field in which the weight of an object is significantly reduced compared to its weight at rest on Earth. When orbiting Earth, a spacecraft is in a condition of continuous free-fall and thus, is in microgravity (<1X10-6g).

2) Realtime downlink - The process of transmitting data (as it is generated) from the orbiter (250-km altitude) via a TDRS (Tracking and Data Relay System) satellite to NASA ground stations. TDRS satellites are positioned in geosynchronous orbit (37,000-km altitude) and provide downlink coverage for approximately 75 minutes of each 90-minute shuttle orbit of the Earth.

3)To be placed on the mailing list for NASA Research Announcements (NRA's) and Announcements of Opportunity (AO), contact:

National Aeronautics and Space Administration
Office of Life and Microgravity Sciences and Applications (OLSMA)
Mail Code UP
Washington, DC 20546-0001.

References

  1. Kennedy Space Center (1991). Information Summaries: Countdown! NASA Launch Vehicles and Facilities, [PMS 018-B (KSC)]. Kennedy Space Center.

  2. Souza K., R. Hogan, and R. Ballard, editors (1993). Life Into Space - Space Life Sciences Experiments - 25 Years: 1965-1990 (Comments Edition). NASA Ames Research Center: Moffett Field, CA, p. 29-89.

  3. Souza K., R. Hogan, and R. Ballard, editors (1993). Life Into Space - Space Life Sciences Experiments - 25 Years: 1965-1990 (Comments Edition).: NASA Ames Research Center: Moffett Field, CA, Appendix III.

  4. Ballard, R.W. and J.P. Connolly (1990). U.S./U.S.S.R. Joint Research in Space Biology and Medicine on Cosmos Biosatellites. The FASEB Journal 4(1):5-9.

  5. Ballard, R.W. and R.C. Mains (1990). Fundamentals of Space Biology. M. Asashima and G.M.Malacinski, eds. Springer-Verlag: New York , p. 21-41.

  6. Bjurstedt, H. (1992). Gravitational physiology in the 1990s. The Physiologist 35(1):S5-S11.

  7. Bonting, S.L., J.S. Kishiyama, and R.D. Arno (1991). Advances in Space Biology and Medicine. S.L. Bonting, ed. JAI Press: Greenwich, CT, p. 279-325.

  8. Dempsey, C.A. (1985). Air Force Aerospace Medical Research Laboratory: 50 Years of Research on Man in Flight. U.S. Government Printing Office: Wright Patterson AFB, p. 1-26.

  9. Dickson, K.J. (1991). Summary of biological spaceflight experiments with cells. ASGSB Bulletin 4:151-260.

  10. Engel, L.A. (1991). Effect of Microgravity on the Respiratory System. Journal of Applied Physiology 70:1907-11.

  11. Grigoriev, A.I. and A.D. Egorov (1992). Advances in Space Biology and Medicine. S.L. Bonting, editor. JAI Press: Greenwich, CT: JAI Press, p. 1-42.

  12. Grindeland, R.E. (1990). Cosmos 1887: Science Overview. The FASEB Journal 4(1):10-15.

  13. Grove, J.L. (1992). Animal Enclosure Module (AEM) Crew Training Familiarization Manual. NASA Training Manual (CT-080) :1-18.

  14. Lange, R.D., L.A. Gibson, T.B. Driscoll, Z. Alleban , and A.T. Ichiki (1994). Effects of microgravity and increased gravity on bone marrow of rats. Aviation and Space Environmental Medicine 65:730-35.

  15. Lanzerotti, L.J. (1990). U.S. space research programs: future prospects. Aviation and Space Environmental Medicine 61:1052-57.

  16. Mazzaschi, A. (1990). NASA's space life sciences program set for major growth in the decade ahead. The FASEB Journal 4(1):3-4.

  17. McDonough, T.R. (1987). Space: The Next Twenty-Five Years. D. Soebel, editor. John Wiley & Sons: New York, p. 9-30.

  18. McDonough, T.R. (1987). Space: The Next Twenty-Five Years. D. Soebel, editor. John Wiley & Sons: New York, p. 31-43.

  19. Meehan, R.T., L.S. Neale, E.T. Kraus, C.A. Stuart, M.L. Smith, and N.M. Cintron (1992). Alteration in human mononuclear leucocytes following space flight. Immunology 76:491-97.

  20. Musacchia, X.J., J.M. Steffen, R.D. Fell, and M.J. Dombrowski (1990). Skeletal muscle response to spaceflight whole body suspension, and recovery in rats. Journal of Applied Physiology 69(6):2248-53.

Acknowledgement - Portions of this work were completed under the auspices of the Penn State Center for Cell Research, a NASA Center for the Commercial Development of Space (CCDS), Grant # NAGW 1196.

This article appeared in the Animal Welfare Information Center Newsletter, Volume 6, Number 2-4, Winter 1995/1996

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