Forget snakes on a plane—how about fish on a spaceship?
Scientific Reports’ December 2016 issue showcases research from Tokyo Institute of Technology on “real-time imaging and transcriptome analysis of medaka fish aboard a space station.” Lead researcher Dr. Akira Kudo says the findings are key to analyzing the impact of microgravity on humans aboard long-term space missions. Kudo and team found, via live-imaging, that “transgenic lines cause immediate alteration of cells which are responsible for bone structure and formation.” The “fish launch” happened in 2014.
It’s well-established that space travel, and the resulting minimized gravity environment, can lead to long-term and sometimes permanent effects on the human body. In fact, the Whole-Body Vibration (WBV) machine that’s popular at many big box gyms was originally designed for astronauts in the 1960s to counteract the gravitational impacts of space travel. NASA’s official site points out that without gravity, muscle and bone density can “weaken alarmingly. Muscles atrophy relatively quickly, while bones lose mass during prolonged exposures to weightlessness.”
Astronauts are required to work out—a lot. Around two hours each day in space are dedicated to working out via unique devices using elastic, harnesses (like TRX workouts) and springs to offer resistance in space like body weight. “Unfortunately, such ‘countermeasures’ have not solved the problem of muscle or bone loss. It’s an ongoing problem for astronauts, and researchers!” says NASA’s government site.
Kudo tackled a big issue with this space mission bone mineral density research. Researchers already know that astronauts have a big reduction in BMD (bone mineral density), but the exact molecular mechanisms in charge of these changes were unknown.
Something’s Fishy
Scientists hand-carried the medaka fish eggs from Japan to Baikonur, Kazakhstan. Medaka, also called Japanese rice fish or Japanese killifish, are small fish common in Southeast Asia and often found in rice paddies (thus the name), marshes, and ponds. Medaka were blast off on the Soyuz rocket launch from the Baikonur Cosmodrome in Kazakhstan, and ensuring the medaka larvae would be healthy in space required careful transportation. Two adult medaka fish were nurtured in Moscow, and extra eggs were collected in case the rocket launch was delayed.
In vivo imaging required hatching the larvae in a custom gel within the fish chambers. Fortunately, there were no major snags in the experiment and the rocket launched as planned. Kudo’s team captured 273 images with 5x objective lenses during this process. Using the “tiling method, ” a complete picture of the medaka chambers was taken.
In collaboration with other researchers in Japan and around the world, Kudo captured remote live-imaging in real-time of fluorescent signals from osteoclasts and osteoblasts of the medaka fish. The fish were only on board the International Space Station (ISS) for one day in microgravity before Kudo captured the first images. Already, Kudo found a spike in osteoclast and osteoblast specific promoter-driven green fluorescent protein (GFP) and DsRed signals. Similar increases continued for eight days in some cases. Exact results varied slightly from fish to fish.
Kudo utilized four double medaka transgenic lines pinpointing up-regulation of the fluorescent signals within osteoclasts and osteoblasts. This clarified the gravitational effect of interactions between osteoclast and osteoblast. Researchers also looked at shifts in gene expression within the fish via transcriptome analysis.
The researcher’s findings suggest that microgravity causes an “immediate dynamic alteration of gene expressions in osteoblasts and osteoclasts.” Specifically, Kudo’s real-time imaging may potentially create a brand new scientific research niche called “gravitational biology.”
A Method to the Medaka
Kudo and his team monitored the ISS-based medaka fish from the Tsukuba Space Center in Tsukubu Science City, Japan. They used an “inverted vertical illumination fluorescence microscope (DMI6000B, Leica Microsystems) partially modified to fit the space environment. All microscope operations on the stage, the object lens revolver, the fluorescence filter turret, and the capacitator were controlled electronically.” Live-imaging osteoblasts revealed how “intense the osterix- and osteocalcin-DsRed in pharyngeal bones” increased in just one day. Osterix- continued to increase for an average of eight days while osteocalcin continued to increase for an average of five days.
TRAP-GFP and MMP9-DsRed fluorescent signals “increased significantly” on days four and five for osteoclasts. By pairing transcriptome analysis with fluorescent analysis to “manage gene expression, ” researchers found that, “HiSeq from pharyngeal bones of juvenile fish at day 2 after launch showed up-regulation of 2 osteoblasts- and 3 osteoclast-related genes.” The nucleus transcription also showed “significant enhancement” via “whole body gene ontology analysis of RNA-Seq.” Researchers noted transcription-regulators were more up-regulated on day two compared to day six.
Five genes were identified as “all up-regulated in the whole-body on days two and six, and in the pharyngeal bone on day two:” c-fos and jun-b, pai-1 and ddit4, and tsc22d3.
From Medaka to Man: Lessons From the “Space Fish”
How do Kudo’s findings impact human space missions? Space’s microgravity environment, where gravity’s force is vastly less than Earth’s, can lead to severe and sometimes permanent problems in the human body. BMD reduction can lead to significant skeletal issues. Calcium loss begins after just ten days in space according to findings in Skylab Flights.
Although more research is needed, Kudo’s team made great leaps in figuring out the exact mechanisms driving bone structure changes in microgravity. Identifying when bone loss begins in the medaka might drive research behind bone loss timelines in space-residing humans. Knowing that shifts in osteoclasts and osteoblasts happen quickly in medakas post-launch can inform future research.
Kudo says the next step is “clarifying the role of glucocorticoid receptors (GRs) on cells in microgravity.” At the Tokyo Institute of Technology, the Japanese “monotsukuri” philosophy is embraced, a combination of innovation and technical ingenuity.
NASA’s Current Bone Loss Prevention Program
In addition to exercise programs, Hiroshi Ohshima of the NASA Space Biomedical Research Office says NASA depends on prophylactic use of bisphosphonate to minimize BMD loss in space. NASA’s approach mimics best practices for preventing BMD loss in the elderly. Ohshima says astronauts work out six days per week totaling up to 15 hours per week while in orbit to prevent BMD loss and kidney stones (another unfortunate side effect of life in space).
However, no number of workouts can stop BMD loss in astronauts. Ohshima estimates that astronauts experience bone mass density loss that’s “ten times that of osteoporosis.” Proximal femur bones lose 1.5% mass per month or about 10% during a six-month orbit. It takes up to four years back on Earth to recover that loss—and that’s assuming a regimented, regular weight-bearing exercise program.
Bisphosphonate is a popular therapeutic agent for osteoporosis, and offers an increase in bone mass and reduction of bone fractures in the elderly. “Through 90-day bed rest research on Earth, we confirmed that this agent has a preventative effect on the loss of bone mass, ” says Dr. Toshio Matsumoto of Tokushima University, a principal investigator of a different study utilized by NASA to prevent astronaut BMD loss.
Dr. Matsumoto recruited participants from NASA and JAXA (Japanese Aerospace Exploration Agency). In collaboration with Dr. Adrian Leblanc of the USRA, Dr. Matsumoto required participants to take bisphosphonate once per week while in space. Although more research is needed, “early results suggest that astronauts can reduce the risk of bone loss and renal stones by proper intake of appropriate nutrients, such as calcium and vitamin D, an effective exercise program, and minimal amounts of medication.”
Currently, preventing BMD loss includes a trifecta of nutrition, exercise, and medicine. Kudo’s findings in the gravitation biology field may influence future studies on BMD loss in astronauts—as well as those on Earth.
