Mitsubishi Heavy Industries Technical Review Vol. 48 No. 4 (December 2011)23

New Development of Space Utilizationin the Life Sciences Field

TOSHIMASA OCHIAI*1 HIROCHIKA MURASE*2

ATSUKO HOMMA*2

JUNICHIRO GYOTOKU*2

Mitsubishi Heavy Industries, Ltd. (MHI) has conducted parabolic flight experiments toassess the potential of life science studies in space and has investigated the impact of microgravity
environments on small animals and humans at the molecular level. The possibility of spaceenvironments contributing to the establishment of new central nervous system pathological models
has been confirmed. Conducting experiments using small animals in space and creating
opportunities for their utilization can be expected to improve our lives because the in-spacescientific data thus provided may help us to understand pathological mechanisms that cannot be
obtained on the ground. This report describes our research projects that will enable theperformance of small animal experiments in space.

|1. Introduction
Using space shuttles, Japan started conducting life science experiments in space in the early
1990s. Since then, MHI has been engaged in the development of experimental equipment. The needfor in-space experiments on small animals, especially mice, is high in Japan, as well as in othercountries. However, these experiments have previously been conducted exclusively by NASA andRussia. As in experiments performed on the ground, mice and rats are indispensable to theunderstanding of biological mechanisms. In the field of space medicine, test results, including
in-space experiments, demonstrate that microgravity environments can affect vital reactions in thebones, muscles and cells of mammals. In fact, major pharmaceutical company in the U.S. haverepeatedly invested money to conduct in-space experiments, but none of the results have been
disclosed to the public. This, in itself, indicates the importance of conducting in-space experiments.
In response to this situation, MHI has focused on research in the drug discovery field with
objectives such as the provision of laboratory animals that have been placed under microgravity
environments, and the establishment of new pathological models. Our research projects have been
conducted based on two different perspectives: 1) the assessment of the impact of microgravity
environments on biological mechanisms, and 2) the establishment of technological backgrounds fora platform for in-space experiments. As far as the former is concerned, we performed themicrogravity experiments on small animals by parabolic flight and verified the effectiveness of theexperiments based on evaluations by universities and pharmaceutical companies. With regard to thelatter, we conducted research to establish a recoverable capsule-type experiment system that can be
piggybacked as a payload on the H-IIA rocket. The component technologies for the maintenance ofan appropriate habitat for mice were examined. Tests related to the closed environment andenvironmental resistance were also performed using mice. Results of these studies have confirmed
the feasibility of the tested technologies. In addition to the system mentioned above, research is
being conducted to develop any possible system that can facilitate the realization of in-spaceexperiments.
*1

Manager, Space Technology Application Department, Space Systems Division, Aerospace Systems*2

Space Technology Application Department, Space Systems Division, Aerospace Systems

Mitsubishi Heavy Industries Technical Review Vol. 48 No. 4 (December 2011)24|2. Impact of Microgravity Environments on Animals and Its
Utilization
Previous studies on space medicine and in-space animal experiments have demonstrated thatmicrogravity environments can affect, within a relatively short time, a variety of biologicalreactions in the mammalian body such as bones, muscles, the cardiocirculatory system, immune
system, and central nervous system. However, the details of individual mechanisms are still
unknown. MHI has repeatedly conducted different basic experiments, and considered thepossibility of drug discovery studies related to the provision of laboratory animals that have beenplaced under the influence of microgravity environments, the establishment of new pathologicalmodels, etc. The following is a discussion of the results showing the effectiveness of microgravity
environments created during the experiments on stress associated with gravity change, including
those of parabolic flight experiments using aircraft.

2.1

Experimental hypothesis

The involvement of serotonin, a neurotransmitter, in the sophisticated control of emotions inanimals (mammals, in particular) and in the development of mental diseases has been
demonstrated. According to previous studies conducted by co-researchers, serotonin is associatedwith the occurrence of panic induced by sudden exposure to microgravity, which is an unknown
environment to mammals. Corticosterone, a stress marker in mouse blood, is significantly elevatedafter the experience of eight successive parabolic flights, as compared to an onboard control group.
This preliminary experiment, therefore, demonstrated that mice were stressed by eight successive
gravity changes (including microgravity). Mice became free from stress three hours after the firstparabolic flight, as they got over the influence of acute stress. To understand the details of thisphenomenon associated with stress, we conducted gene expression analysis considering serotonin
as the key molecule.

2.2

Gravity change-associated stress experimental system

A parabolic flight with MU-300 can create a microgravity field of approximately 10-2 g for
20 seconds at most. In a single flight, a parabolic flight can be repeated a maximum of 20 times. In
this experiment of stress associated with gravity change, an aircraft was carefully maneuvered; theapplied gravity during the control of aircraft orientation before/after each parabolic flight wasmaintained at (or below) 1.4 g to avoid hyper gravity as much as possible. The duration of
microgravity achieved in a single parabolic flight was approximately 15 seconds. Gradual change
in aircraft orientation allowed this microgravity flight to be repeated eight times within the overall
aircraft flight time of 1 hour. This flight method is hereafter referred to as the gravity
change-associated stress experimental system (Figure 1).

Figure 1

2.3

Image of an aircraft experiment

Experimental method

In this experimental system, based on the data obtained from previous basic experiments, thecontrol group was defined as the on-board group treated immediately before the first parabolic
flight. The experimental group was defined as the group treated six hours after the first parabolic
flight (Figure 1). Features of this experimental system include the capability of conducting various
on-board treatments, such as blood or tissue sample collection from the experimental animals
before/after each parabolic flight, if necessary. In this experiment, mouse blood and brain/small
intestine tissue samples were collected on-board to perform genetic analyses.

Mitsubishi Heavy Industries Technical Review Vol. 48 No. 4 (December 2011)25

2.4

Results

Of serotonin-related genes, Tph1 is associated with tryptophan hydroxylase, which acts on
the peripheral nervous system, such as the small intestine. Tph2 is related to another form of
tryptophan hydroxylase that acts on the central nervous system such as the brain. In thisexperiment, only the Tph2 level was significantly elevated, exhibiting increased gene expression in
the serotonin synthesis system (Table 1).
Table 1Analysis results of stressors associated with gravity change and acrophobia
Gene name

Functions

Serotonin synthesis system(peripheral nervous system: small
intestine)
Tryptophan
Serotonin synthesis system
Tph2hydroxylase(central nervous system: brain)
Monoamine oxidase Serotonin degrading system
Maoa
A(substrate: serotonin)
Monoamine oxidase Dopamine degrading system
Maob
B(substrate: dopamine)
Serotonin transport
Sert
Serotonin transporter(for recycling)
Large neutral amino Tryptophan
Slc7a5acid transporter 1
Htr1a
Serotonin receptor Serotonin receptor protein
Tyrosine
Noradrenaline, dopamineThhydroxylase
nervous system
Glutamic acid
Gad1(67kDa)
GABA nervous system
decarboxylase 1
Glutamic acid
Gad2(67kDa)
GABA nervous system
decarboxylase 2
Tph1

*１

Tryptophanhydroxylase

Gravitychange*1

Acrophobia*1

Socialsituation

－

－

ND

－

－

－

ND

－

ND

－

－
－
－

－

ND
－

－

ND

－

ND

－

ND

Red arrow:pertaining to the brain (with significant change)
green arrow:pertaining to the small intestine (with significant change)
purple arrow:pertaining to the brain (tendency, without significant change)－ horizontal line:representing “no change”
ND:representing “not done” (based on references)

Note : The RNA expression level was quantitatively estimated from real-time polymerase chain reaction (PCR)
with the intercalator method, SYBR Premix Ex Taq II, where synthesized cDNA of the target andhousekeeping genes was used as a template. HA067799 (18SrRNA) was selected as the housekeeping
gene.

Conversely, because Tph2 expression is not accelerated by acrophobia or social stress, theserotonin synthesis system in the midbrain was stimulated by the stress caused by gravity change.
Maoa is a gene related to a serotonin-degrading enzyme, while Maob is a gene associated with adegrading enzyme acting on another substrate, dopamine (also a neurotransmitter). The
experimental results indicate that the stress associated with gravity change enhanced only Maob
gene expression (dopamine-degrading enzyme), facilitating dopamine metabolism while selectively
retaining serotonin levels. The expression of Sert, which is involved in the recycling pathway
related to the effective use of synthesized serotonin, was enhanced by gravity change, but was
lowered by acrophobia-induced stress. Slc7a5 functions as a large neutral amino acid transporter totake tryptophan into the cells. The increased gene expression of Slc7a5 also indicates thefacilitation of the serotonin synthesis system. The increased expression of Htrla, a synthesized
serotonin receptor, was seen only under acrophobia-induced stress.
Tyrosine is an amino acid from which other neurotransmitters such as adrenaline,
noradrenaline, and dopamine are produced. Th is a gene associated with a tyrosine synthesis
enzyme that is involved in the first stage of tyrosine synthesis. On the other hand, GABA is aneurotransmitter that inhibits brain excitation. Gad1 and Gad2 are the genes that are associated withGABA as glutamic acid decarboxylase. Of these three, Th showed a tendency for enhanced
expression, which indicates that the stress caused in this experiment increased the expression of Th
in the brain. Therefore, the tendency to activate the entire nervous system by facilitating theactivation of dopamine, noradrenaline and adrenaline systems was observed. However, thistendency did not reach statistical significance.

Mitsubishi Heavy Industries Technical Review Vol. 48 No. 4 (December 2011)26

2.5

Discussion

In the conventional methods for stress induction used in studies on mental diseases such as
the elevated plus maze, the release of not only serotonin but also other amino acids including
dopamine and GABA in the brain, is accelerated. Therefore, it was not possible to selectively
facilitate the expression of serotonin synthesis genes. The results of the gene expression analysis in
the midbrain or small intestine showed that the gravity change associated-stress experimental
system was a new stress induction method by which the serotonin synthesis system can beselectively stimulated in response to stress. Use of this new experimental system in research maylead us to the discovery of new pathological mechanisms in the central nervous system.

|3. New Experimental Platform
MHI has developed space experiments in the life science field, including the cell biology
experiment facility (now in operation) on the International Space Station (ISS), which is in orbit.
Based on this experience, MHI has conducted research related to a recoverable capsule-type
experiment system as a means of realizing the utilization of space for drug discovery since 2004.
This system is mounted on the H-IIA rocket as a piggyback payload and can offer an experimental
platform for biological experiments from launch until recovery. In the development of the system,
basic data in terms of metabolism and behavior of small animals, etc. were examined to determine
the conditions for small animal habitat in space. To realize such conditions, component
technologies were developed and their verifications were conducted. Thus, we have accumulated
technologies that can serve as an infrastructure for small animal experiments.
If this system becomes available for practical use, space can be utilized as a newexperimental site for small animal experiments. This may enable not only the discovery of
biological findings from small animals used in in-space experiments but also the use of such asystem as a platform for drug discovery.

3.1

Our original technology for small animal experiments

The biological experimental system using the H-IIA rocket offers an experimental
environment starting from the launch of small animals (mice) into space until their return to theground. The system consists of a satellite bus (transportation infrastructure) and an experiment unit
(infrastructure for small animal experiments). The experiment unit is equipped with sub-systems
for environmental control and life-support functions and other experimental functions (Figure 2).
Environmental control and life-support
functions
・ Functions as a habitat(securing the floor area)
・ Functions to control environmental
factors(maintenance/management of
temperature, humidity, CO2 level, O2level, and trace gas/odorous component
levels)・ Life-support function(food and water supply)・ Waste management(disposal of urine and feces, in particular)

Other experimental(habitat) functions
・ Habitat environment(management of noiseand light)
・ Monitoring function(collection ofenvironmental andimage data)

Figure 2 Schematic diagram of the piggybacked recoverable capsule-type experimental
systemMitsubishi Heavy Industries Technical Review Vol. 48 No. 4 (December 2011)27

For realization of the above functions and conditions, it is important to overcome thetechnical challenges resulting from the distinctive features of in-space experiments, such as
microgravity (space with no sense of direction), change in wettability of a liquid and environmental
change during launch or re-entry (Table 2).
Table 2 The technical challenges and corresponding suggestions for technical solutions
Major technical challenges
peculiar to the environment in
Suggestion for technical solution
Demonstration statusspace
For habitation Necessity of securing a suitable Adoption of a habitat type with a ・ 34-hour closed
habitat size according to the doughnut shape (a loop of corridor) to environment test,
number of mice aboard and create the least restriction of activities performed
shortening the length of air flow among mice and to shorten the length (Number of mice: 3)
inside the habitat to prevent of air flow, which has been realized ・ 2-week closed
cross-contamination of floating (Figure 3).
environment test,
waste to mice.
performed(Number of mice: 5)
Waste disposal Necessity of preventing the Use of absorptive walls to immediately ・ Waste-disposal
diffusion of urine onto the mouse absorb liquid waste (urine, in component test,
body surface or the attachment of particular) and adoption of air performed
feces to mice.
circulation to collect solid waste at the (Parabolic flightend of air flow, which has been realized experiment with an(Figure 3).
aircraft)Environmental Necessity of removing the Adsorbent
andvalve-control ・ 34-hour closed
control
influence due to the change of technology to achieve a closed type of environment test,
experimental environment (e.g., environment control with the external performedpressure change on launch, and influence being shut out from the (Number of mice: 3)change in air composition, mouse habitat environment (removal of ・ 2-week closed
temperature and humidity due to CO2 and harmful gases, and control of environment test,
metabolism of astronauts, etc.) O2 supply), which has been realized. performed
and preventing the contamination With regard to a type of habitat with an (Number of mice: 5)of experimental environment air vent connected to the outside ・ Deodorizing effect(especially, contamination of (residential area of astronauts, in verification
test,
habitatenvironment
by particular), adsorbent and filtering performed
astronauts).
technology to keep the concentrationsof trace gases and odorous components
in the habitat at desirable levels forhabitation, and to prevent their leakage
into the outside, has been developed.
AnimalNecessity of receiving the least Automatic animal fixation technology ・ Verification test for
fixation
influence of disturbances such as using gas displacement and cooling, animal fixation
hypergravity created before/after which has been realized. New characteristics/
the microgravity experiment, experimental method by which quality of fixed
because these parameters are samples
are
fixed
before/after tissue, performed
considered uncertain in the microgravity without the influence ofassessment of results.
disturbances,
which
has
been
established (Figure 3).
Function

The functions for habitation and waste disposal are closely related to each other. From a
habitation viewpoint, a sufficiently large space should be secured so as not to constrain theactivities of small animals. On the other hand, a smaller space is preferable from a waste-disposal
point of view, because small animal urine and feces can be removed before the waste, especiallyurine, spreads over the body surface (prevention of cross-contamination). In response to thefunctional requirements that contradict each other, we developed the component technologies tooptimize factors such as the habitat shape, air flow inside the habitat, and waste disposal method.
The closed environment test, in which the environment inside the habitat was controlled without
the influences of external environment, and the adaptability test to microgravity environments byparabolic flights were conducted. Thus, the verification of each component technology has been
performed (Figure 3).
In the waste disposal method screening test, mock mice with an imitation body surface were
made. When the microgravity experiment started, the mock mice automatically started to excrete
mock urine that was also created using a dyed liquid with viscosity and surface tension equivalent
to the real liquid waste (urine) of mice. The behavior of mock urine in terms of spreading over themouse body surface was examined. The results show that the passive adsorption method was
effective for the disposal of liquid wastes from a short-term perspective, while the active adsorption
method should be adopted for a long-lasting effectiveness.
To enable the evaluation of the impact of environmental change before/after the in-spaceexperiment (gravity change, in particular) on small animals, MHI has also made the animal fixation
technology available for practical applications. Figure 4 shows an example of the results of theassessment test regarding the whole-body fixation of mice with our animal fixation device.

Mitsubishi Heavy Industries Technical Review Vol. 48 No. 4 (December 2011)28

Figure 3

Functional verification tests for habitation and waste disposal

Figure 4

Animal fixation technology test

With this device, the air inside the mouse habitat can be displaced by carbon dioxide gas
within a short time, enabling the treated mice to be kept refrigerated. In the assessment test, 10
week-old male mice were treated with carbon dioxide gas before being kept either refrigerated or atroom temperature. Protein, RNA, and a transverse muscle slice samples were then removed fromthe mouse anterior tibial muscle to compare the fixed samples according to the preservation
method. As a result, the presence of differentially-localized muscle fibers, as seen in the damaged
muscles, was observed in the samples kept at room temperature. On the other hand, in the samples
refrigerated after the animal fixation, the condition of fixed samples was preserved normally. The
effectiveness of the device has been sufficiently demonstrated.

3.2

Toward more advanced small animal experimental devices

The microgravity environment created by a parabolic flight only lasts a number of seconds.
However, the use of accumulated technical knowledge with regard to small animal experiments
will enable experiments conducted by suborbital flights, in which microgravity duration reaches
several minutes, or even the development of small animal experimental devices with which an
experiment can be conducted on the ISS for several months.
Particularly when it comes to the devices used on the ISS, only minimum life-support
functions (e.g., food and water supply, waste disposal, and ammonia removal) are required as basic
specifications because the environment control functions equipped in the ISS can be utilized.
Depending on the objective or needs of each small animal experiment, it is also possible to adopt asystem in which the functional components can be replaced as cartridges, to achieve lowered CO2
levels, and/or to be equipped with an animal monitoring system or animal fixation device. An
operational scenario for the use of small-animal experimental devices on the ISS is shown in
Figure 5. In this scenario, the restrictions on the timing for late access and the on-board weight,
depending on the means of transportation, are considered. The device consists of three components:
the main body, a cage in which mice are reared, and spare goods (cartridges). The device itself will

Mitsubishi Heavy Industries Technical Review Vol. 48 No. 4 (December 2011)29

be on board the ISS until all experiments are finished. A cage with mice inside will be replaced
according to the experiment that is to be conducted. Cartridges will be replaced regularly, based on
the schedule of experiments. Thus, the continuous and effective performance of experiments withdifferent objectives will be possible. Looking further ahead, we will develop an analysis device
with which the fixed samples can be examined while they are still in orbit. The combined use of theanalysis device and small animal experimental devices will shorten the time necessary forexperimental assessment. We expect that we can expand the number of possible users of small
animal experimental devices on the ISS.

Figure 5

Scenario of in-orbit demonstration|4. Conclusion
Through the development of equipment for life science experiments, MHI has focused on
microgravity impact on the body and has conducted research to utilize space as a platform creation
for drug discovery. We will deepen our understanding through the verification of the effectivenessand assessment of microgravity impact on the body. With this in mind, to provide small animals
that have been placed under the influence of microgravity environments, we will continue theresearch projects and establish the infrastructure for in-space experiments on the ISS or bysuborbital flights. This will create potential new clients such as universities, research institutes andpharmaceutical companies, which may contribute to active space utilization.

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2.

Yoshioka, M. et al., Parabolic flight-induced serotonin related gene expressions in mice. Folia
Pharmacologica Japonica Vol.135 No.3(2010)
Matsumoto, H. et al., The Feasibility of Utilizing Space in Drug Discovery, Mitsubishi Heavy Industries
Technical Review Vol. 45 No. 4 (2008) pp.50-54