Effects of Simulated Hypergravity on Biomedical Experiments - PUCRS

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Effects of Simulated Hypergravity on Biomedical Experiments

THAIS RUSSOMANO, MARA R. RIZZATTI, RODRIGO P. COELHO, DIOGO SCOLARI, DANIEL DE SOUZA, PAULA PRÁ-VELEDA

his paper presents the conception and development of a centrifuge for hypergravity research use. Experiments were recorded via a digital camera and the images acquired were processed for better visualization of the effects of simulated variation of gravitational force on models of living systems and plant germination. Four different test models were used to illustrate the effects of 1.5 Gz and 7.0 Gz on human physiology, including the cardiovascular and musculoskeletal systems. One experiment was conducted to evaluate plant germination and growth after intermittent hypergravity exposures to 7.0 Gz, which can demonstrate the usefulness of centrifuges on plant development in extra-terrestrial colonies.

T

Keywords: hypergravity, centrifuge, human physiology, biomedical experiments, space physiology

Introduction

Konstantin Tsiolkovsky, a pioneering Russian rocket scientist, once said that “The Earth is the cradle of Humanity, but we can not live in a cradle forever.” Mankind in its process of evolution will eventually leave its home planet searching for new places to inhabit, just like our primitive ancestors did when they left the cave. Living organisms on Earth share at least on thing in common: the fact that they all live under the influence of the gravitational force of the Earth, which produces an acceleration of 2

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approximately 9.8 1m/s2 at mean sea level (indicated by the symbol g). Any variation in this force should result in physiologic changes in any given organism (13). Gravity was first described by the English physicist Sir Isaac Newton more than 300 years ago, as the attraction force between any two masses. It is one of the four basic forces in nature, the others being the electromagnetic force, strong nuclear force and the weak nuclear force). The AustrianCzech physicist and philosopher Ernest Mach and the German physicist Albert Einstein concluded that gravitation and acceleration forces are equivalent, and produce the same effects on all bodies whatever their nature (7). In space missions, astronauts are exposed to either microgravity in space or hypergravity during launch and reentry. During hypergravity, the resultant acceleration in a body is different from the acceleration due to the Earth’s gravity. The unit of the ratio of an applied acceleration to the gravitational constant is conventionally called G, and calculated by the expression (1): G=

applied acceleration g

(1)

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Therefore, if the resultant force is 2G, the applied acceleration will be 19.62 m/s2 divided by 9.81 m/s2 . Gell (4) first introduced the concept of human body G vectors in 1961, using an axial nomenclature system that has been the basis for studies related to acceleration physiology since then. The three major axes are longitudinal (Z), lateral (Y) and horizontal (X). The direction of acceleration forces along the axes is referred to as (+) or (−). The inertial forces are opposite to the acceleration forces, as indicated in Figure 1 (Burton et al, 1985).

−GZ

−GY

−GX

For research and pilot training, it is possible to create on the ground, hypergravity environments similar to those commonly found in a space mission or during a steep maneuver of a high performance aircraft for research and pilot training. Human centrifuges have been widely used around the world for these purposes, especially to study and to demonstrate the effects produced on the cardiovascular system during exposure to different +Gz acceleration profiles. Terrestrial animals, including humans, require regular periodic gravitational stimulation to maintain terrestrial physiologic functions on Earth or in space. Using a centrifuge, G simulation equivalent to that of Earth can be produced in space (14). By increasing the levels of G above 1 G, such simulation may be more inexpensive, efficient and effective in preventing the physiologic deconditioning in space than many other countermeasures not utilizing artificial gravity (12). The use of periodic, increased G exposures in space referred to as artificial gravity (5) may offer a possible countermeasure to physiologic deconditioning in-flight. Theoretically, artificial gravity can be expected to be more comprehensive in its effect on human physiology, acting on more than one body system as it does, than countermeasure interventions currently used. Centrifugation has also been used widely in the separation of cellular components, but only occasionally as a primary environmental stimulus. Edwards and Gray (1977) performed a series of studies looking at the effect of hypergravity simulation on cells and E. Coli. In the range of 2 Gz to 25 Gz, Auxin transport and geotropic response in coleoptiles (the protective sheath around the embryonic shoot in grass seeds) are increased and growth seems to be stimulated. Above that, however, damages have been observed both in cells and E. Coli. From 25 Gz to 500 Gz, coleoptile growth is reduced and some morphological changes may be seen. At 1000 Gz to 2500 Gz, root formation in willow cuttings increases. From 1000 Gz upward, cytoplasmic stratification occurs and seed germination decreases. Between 200 Gz and 15000 Gz, chromosome damage has been observed. Algal cell polarity may be reversed at 5000 Gz to 20000 Gz. Above 30000 Gz, the response of some cells to gibberellic acid is halted. Permanent morphologic changes in Escherichia coli are produced at 110000 Gz. Some plant cells have survived 176000 Gz for 20 hr (3). This study aims to present the conception and development of a centrifuge for hypergravity research use. Experiments were recorded via a digital camera and the images acquired were processed for better visualization of the effects of simulated variation of gravitational force on models of living systems and plant germination. Four different test bodies were used to simulate the effects of 1.5 and 7.0 Gz on human physiology, including the cardiovascular and musculoskeletal systems. One experiment was conducted to evaluate plant germination and growth after intermittent hypergravity exposures to 7.0 Gz, which can demonstrate the usefulness of centrifuges on plant development in extra-terrestrial colonies.

+GX

+GY

Materials and Method +GZ Fig. 1. Standard acceleration nomenclature. The arrows indicate the direction of the inertial reaction to an equal and opposite acceleration.

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The Centrifuge for Simulated Hypergravity Experiments

A small centrifuge was conceived and developed by the Microgravity Laboratory/IPCT-PUCRS for the performance MARCH/APRIL 2007

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Micro-Camera

Arm Test Body and Accelerometer Support Base

Motor and Gearing System

Power Supply

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Fig. 2. Centrifuge with test body at rest.

Ny

Axis

Arm

Test Body

W

Ny

N

Axis

long arm. The motor is connected to the center of the arm, dividing it into two equal arm lengths of 600 mm, thus allowing two experiments to be conducted at the same time. The test model is placed at the end of each arm, along with a digital camera for image acquisition during rotation, and an accelerometer, used to indicate the level of acceleration achieved in an experiment. The final gear relation of 5:1 is achieved in two steps. The first gear, attached to the motor, is 23 mm in diameter and is connected to another gear with a diameter of 74.75 mm, providing a ratio of 3.25:1. The second gear is connected by an axis to one with a diameter of 32 mm, which in turn is connected to another gear of 49.6 mm, giving a ratio of 1.55:1. The final ratio of 5:1 is obtained by multiplying 1.55 by 3.25. Rotation of the system is controlled by a simple DC power supply that is directly connected to the DC motor. The desired rotation per min (rpm) is established by changing the voltage of the power supply. The acceleration is measured using an ADXL150 accelerometer. The signal is digitalized through an A/D converter and converted to RS232 communication protocol by an ADuC812. The signal is then sent to a computer via radio Radiometrix TX2 UHF transmitter. A Radiometrix RX2 UHF receiver is attached to the computer to receive the data. The motor is able to accelerate the system up to 36 Gz, equivalent to 36 times the force of gravity on Earth (16). The G force was calculated by the equations (2) and (3). The force diagram of the system is represented in Figure 3. The weight (W) acting on a test body at rest is balanced by the normal force (N). During rotation, however, a centripetal force (FCP ) starts acting on the test body pulling it towards the center of the circle, while the inertia keeps the test body in a tangential direction. The resultant force, i.e., the apparent weight (WA ) produced is related to the G effect applied on the test body.

Arm

Inertia

FCP (NX)

Test Body

W

WA

Fig. 3. Schematic view of the physical forces acting on a test body at rest (above) and during rotation (bottom).

of different hypergravity experiments (16). The centrifuge (Figure 2) consists of a motor fixed in a steel base (120 mm height, 350 mm length, 230 mm width) supporting a 1200 mm 4

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W2A = F2CP + W2 G = WA /W

(2) (3)

c b

a

A digital camera with the following specifications was used to acquire the images during rotation: still image capture resolution of 320 × 240 pixels; video capturing resolution of 640 × 480 pixels; video capture speed of 30 frames per second. The camera was compatible with a PC operating system and Windows 98. Test Models

d

Fig. 4. Test Models.

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Four different test models (Figure 4) representing body systems were used to demonstrate the effects of two levels of hypergravity: a sealed system of tubes filled with a colored liquid to simulate a column of blood (A), three magnetic rings representing vertebrae (B), a spring (C) and a rubber band (D) with an 8 g mass placed on the free extremity to simulate a muscle. After the test model was placed at the end of the centrifuge arm, the system was rotated at 38 rpm and 92 rpm to produce 1.5 Gz and 7 G, respectively. Rocket Plant–Eruca Sativa Mill

A plastic container (60 mm diameter and 62 mm height) with 44 g of black sand (Humosolo, Humus type) and 10 seeds of Eruca Sativa Mill (KAD type—humidity proof) was placed at each end of the cen(a) (b) (c) trifuge arms and rotated at 7 Gz (92 rpm) from 8 AM to 5 PM daily (a Fig. 5. The system of tubes filled with a colored liquid at 1.0 G (0 rpm) (a), 1.5 Gz (38 rpm) total of 9 h/day) for 4 consecutive (b) and 7.0 Gz (92 rpm) (c). days. The containers were covered with a plastic lid to avoid evaporation drying of the sand by the generated wind. The container was safely secured to the end of the centrifuge arm through a metal pin. It was placed at 90◦ in relation to the centrifuge arm, remaining at that position at rest. During centrifuge rotation, it moved upwards reaching an angle of 0◦ . One hole of 3 mm (a) (b) (c) was made on the side of each plastic container to allow ventilation. Fig. 6. Three magnetic rings at 1.0 G (0 rpm) (a), 1.5 Gz (38 rpm) (b) and 7.0 Gz (92 rpm) Two similar plastic containers (con(c). trol) kept open in the same room served as control. Room temperature was set at 22◦ C. Water (Figure 5). (0.5 ml) was added to the containers before and immediately This experiment can be used to represent the column of after the experiment. The experiment was performed twice to blood in the human body during different levels of hypergravtest reproducibility. ity exposure. Both the blood in the vascular system and the colored fluid in the capillary system at 1 G are attracted to the Results and Discussion center of Earth, due to the action of gravity. In microgravity, where the resultant force is virtually zero, the blood is uniSystem of Tubes Simulating the Column of Blood formly distributed in the human body. When Gz force At rest, the colored liquid inside the system of tubes was in increases, however, due to the centrifuge rotation, the colored the lower half of the structure. As the Gz increased during liquid is progressively displaced towards the end of the capilrotation, the liquid was displaced partially at 1.5 Gz and comlary system. This is analogous to what happens to the circulapletely at 7.0 Gz towards the far end of the system of tubes tion of a pilot in a fighter aircraft when it suddenly changes IEEE ENGINEERING IN MEDICINE AND BIOLOGY MAGAZINE

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(a)

(b)

(c)

Fig. 7. Spring with a mass at 1.0 G (0 rpm) (a) 1.5 Gz (38 rpm) (b) and 7.0 Gz (92 rpm) (c).

case was complicated by a C6-7 inter-vertebral disk prolapse and a congenitally narrow spinal canal. The second case involved progressive degenerative spinal stenosis in the C5-6 disk space. These two cases suggest that +Gz forces can cause degenerative spinal stenosis of the cervical spine. Flight safety may be jeopardized if symptoms and signs of medullar compression occur during high +Gz stress. Spring and Rubber Band with an Attached Mass Modeling Muscle Function

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The spring (Figure 7) and the rubber band (Figure 8) with an attached mass were increasingly stretched at 1.5 Gz and 7.0 Gz. Numerous observations suggest the importance of gravitational force in regulating muscle mass. (a) (b) (c) Centrifugation is believed to be useful for preventing muscle functional and structural loss under micrograviFig. 8. Rubber band with a mass at 1.0 G (0 rpm) (a), 1.5 Gz (38 rpm) (b) and 7.0 Gz (92 ty conditions (15). Ohira and colrpm) (c). leagues studied the effect of hind direction during a steep maneuver, exposing the pilot to high limb suspension in rats, used to simulate microgravity, or cenGz acceleration. This force can be sufficient to induce a loss of trifugation at 2 Gz between post-natal day 4 and month 3 and consciousness (G-LOC). The physiologic cause of G-LOC is after a 3-month recovery period back in 1 G on the characterisinsufficient perfusion of blood to the brain and subsequent tics of muscle (17). Pronounced growth inhibition was induced hypoxia. If the onset rate of +Gz acceleration is gradual, the by unloading, but not by 2 Gz force. It is suggested that the brain hypoxia shows up first as reduced peripheral vision. development and/or differentiation of soleus muscle fibers are First, a reduction of the peripheral vision (Grey-out) is noted, closely associated with gravitational force. The data showed that followed by a loss of the central vision (Black-out) (6). gravitational unloading during postnatal development inhibits Figures 5(b) and 5(c) can be used to schematically represent the myonuclear accretion as indicated by the subnormal numGrey-out/Black-out and G-LOC, respectively. bers of both mitotic active and quiescent satellite cells. Even though the fiber formation and longitudinal fiber growth were Magnetic Rings Representing Inter-Vertebral Discs not influenced, cross-sectional growth of muscle fibers was also During hypergravity, the magnetic rings were progressively inhibited together with lesser myonuclear DNA content per unit displaced from their resting position at 1 G, decreasing the disvolume of myonucleus. Unloading-related inhibition was genertance between individual rings during rotation (Figures 6). ally normalized following the recovery. Weightlessness and bed rest, a widely used ground-based Akima and colleagues (9) studied the effect of intensive cycle microgravity simulation, reduce the mechanical loading on the training with artificial gravity on the maintenance of muscle size musculoskeletal system and consequently increase the distance during bed rest. Ten healthy men were divided into 2 groups: a between inter-vertebral discs. This effects increases the nutricountermeasure group, BR-CM (n = 5); and a control group, BR-Cont (n = 5 ). The BR-CM subjects undertook intensive tional diffusion distance and alters the mechanical properties of cycle training (to 90% of maximum HR) with short-arm centhe spine. The spinal lengthening is thought to cause back pain trifuge-induced artificial gravity on alternate days during in astronauts (1). The opposite, however, occurs during hyper20 days of bed rest. Muscle volume of the thigh and maximum gravity exposure, as simulated in Figures 6(b) and 6(c) with the voluntary contraction (MVC) during isometric knee extensions three magnetic rings representing inter-vertebral discs. Previous were measured before and after bed rest. Muscle functional magnetic resonance imaging (MRI) studies have shown that magnetic resonance imaging (mfMRI) and electromyogram repeated exposure to +Gz forces can cause premature degener(EMG) of the quadriceps femoris were obtained during subative changes of the cervical spine, as a work-related disease. A maximal knee extension exercises at a load of 30% MVC. The study was conducted by Hamalainen and colleagues in 1999 findings showed that the volume of the total thigh muscles was (12) on two clinical cases of +Gz-associated degenerative cermaintained in the BR-CM group (−1%), whereas it was not in vical spinal stenosis, a condition in which the distance between the BR-Cont group (−9%, p < 0.05 ). MVC decreased in the inter-vertebral discs is reduced due to the action of dorsal osteoBR-CM (7%) and BR-Cont groups (23%). EMG activity in the phytes in fighter pilots. Conventional x-rays and MRI were used BR-CM group after bed rest was significantly lower than to demonstrate narrowing of the cervical spinal canal. The first

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before; however, no significant change was found in the BR-Cont group. There were no significant changes in the resting and exercised mfMRI signals in either the BR-CM or BR-Cont groups. These results suggest that intensive cycle training with hypergravity maintained the size of human skeletal muscles during bed rest.

Lid for the Recipient

Eruca Sativa Mill

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Rocket seeds are expected to germinate within 4 to 7 days. It was observed that the seeds exposed to 7 Gz germinated i n 3 days as compared to 4 days for the control seeds. Individual plant height Hole for could not be obtained during the experiVentilation ment days because measurements had to include the roots. The growing plants Plant Growth After 4 Days Plant Growth After 4 Days Experimental Recipient – 7 Gs were removed at the end of the experiControl Recipient – 1 Gs ment and then measured with a paquimeter from root to top. Two experiments were performed under the Fig. 9. The growth of individual Eruca Sativa Mill after 4 days at exposure to intermitsame conditions. tent 7 Gz and at 1 G (control)—Experiment 1. Mean plant height at 7 G in the first experiment was 3.2 cm versus 1.9 cm of the Control (Figure 9). However, a statistical test could not be US Lab “Destiny”. performed due to the low number of germinated seeds of the The experiments are housed in standard Experiment control (n = 3) in relation to the seeds exposed to hypergravity Containers, allowing either research in microgravity or accel(n = 9). In Experiment 2 (n = 14), mean plant growth of 2.2 cm eration studies with variable G-levels, when mounted on the at 7 Gz was significantly higher than the plant growth at 1 G centrifuges. While Biopack provides only thermal control, (control) of 1.8 cm (p = 0.02). BIOLAB and EMCS supply each container with a dedicated The result of above average growth of the plants after being atmosphere (controlled CO2 , O2 concentrations and relative humidity and trace gas removal): EMCS also contains fresh exposed to hypergravity simulation, as well as the increased and waste-water reservoirs on its rotors. Power and data lines number of germinated seeds found at 7 Gz in Experiment 1, are available in all the described facilities. Highly automated suggests that further studies related to the effect of hypergravity systems, like BIOLAB’s Handling Mechanism and Analysis on plant growth need to be conducted. Instruments, support the tele-science concept and help reduce A theory that might explain the significant growth of the crew time in orbit. A BioGlovebox with its support instruEruca Sativa Mill after 4 days of intermittent hypergravity ments offers unique research facilities in Space. The feasibiliexposure is based on the influence of the Auxins on plant ty of offering experiment hardware inside the containers has growth. been studied by ESA for several kinds of Experiment Support The plant hormone Auxin, a term derived from the Greek, Equipment that could potentially be used for research in which means to grow, is characterized by its capacity to developmental biology; for example in experiments with induce the elongation of cells in the sub-apical region of small eggs, cells and tissues, with small aquatic animals, with branches. Auxin also affects physiologic processes, including insects and with plants (8). phototropism, geotropism, fruit development and gender Centrifuges are particularly important for research in gravitaexpression. However, cellular elongation is one of the most tional biology because the inertial forces developed by motion important effects of the Auxin action. An important step of can be combined with gravitation to produce gravitational fields elongation is the acidification of the cellular border. This is other than Earth’s gravity. In orbiting satellites centrifuges can generated by an electrochemical gradient that leads to proton provide an on-board 1 G environment. They can also be very secretion through the plasmatic membrane, promoting the useful for Mars and Moon colonies to counteract the effects of acidification of the cellular wall. This leads to an increase in hypogravity or microgravity on human and animal physiology enzymatic activity, which promotes the malleability of the cell and plant growth (2)(12). edges that allows cellular elongation. Then the osmotic pressure forces the entrance of water into the cell, causing the Conclusion expansion of the cell (3). A small centrifuge, conceived and developed by the Three new European Space Agency (ESA) facilities will be Microgravity Laboratory/IPCT-PUCRS, was used to perform available for biological experiments in Space, Biopack on the different hypergravity experiments (16). Two different rotaSpace Shuttle and two instruments on the International Space tions were used to simulate 1.5 Gz and 7.0 Gz on test models. Station: BIOLAB in the European “Columbus” Laboratory The experiments succeeded in modeling cardiovascular, musand the European Modular Cultivation System (EMCS) in the IEEE ENGINEERING IN MEDICINE AND BIOLOGY MAGAZINE

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culoskeletal structure and function during hypergravity. In addition, one experiment was conducted to evaluate plant germination and growth after intermittent hypergravity exposure, with a view to examining the potential use of such centrifuges in extra-terrestrial colonies. The study findings suggest that seeds of Eruca Sativa Mill (Rocket) respond well to intermittent +Gz exposure.

The article is about a centrifuge system that can be used to for hypergravity research to see the effects of gravitational force on humans and plants for possible future space travel/colonization and to counteract these effects in space.

Acknowledgment

References

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We would like to thank the Instrumentus Indústria e Comércio Ltda. and the Centro Industrial de Equipamentos de Ensino e Pesquisa (Cidepe), located in Canoas, RS, Brazil, for the technical support and engineering expertise provided for the completion of this work.

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