The Mice Drawer System （MDS） Experiment

特別講演

Ranieri Cancedda^1,2, Roberta Biticchi², Michele Cilli², Delfina Costa1², Yi Liu1², Federica Piccardi¹, Alessandra Ruggiu^1,2, Roberta Tasso^1,2, Sara Tavella^1,2

¹Universita' degli Studi di Genova
²Istituto Nazionale per la Ricerca sul Cancro, Genova
Vittorio Cotronei and Salvatore Pignataro
ASI−Agenzia Spaziale Italiana, Italy

Several organs and apparatus are affected by weightless conditions and in particular by the weightless experienced during space flights.　Therefore space missions are good opportunities to investigate in a whole organism the controlling cellular and molecular mechanisms.　For this type of studies mice are an excellent animal model for several reasons:reduced body size, relatively short time needed to reach adulthood, availability of strains with different genetic background and of different transgenic lines, etc.　In line with the International Space Station （ISS） development, the Italian Space Agency （ASI） contracted Thales Alenia Space Italia, the largest Italian aerospace industry, to design and build a space flight payload for rodent research on ISS:the Mouse Drawer System.　This payload can be integrated inside the Space Shuttle middeck during transportation to/from the ISS, and inside the Express Rack in the ISS during experiment execution.　It is designed to perform experiment as much automatically as possible;only maintenance activities require procedures involving crew.
　The first MDS experiment was launched inside Shuttle Discovery in STS-128 on August 28 2009 at 23:58 EST, and returned to earth by Shuttle Atlantis in STS129 on November 27 2009 at 9:47 EST, marking it as the first long duration （3 months） animal experiment on the International Space Station （ISS）.
　A ground replica of the flight experiment （“ground control”） was performed at the University of Genova from November 2009 to the second week of February 2010, while a parallel experiment by exposure the mice at a 2 G environment （centrifuge） was performed at the graduate School of Medicine of the Osaka from June to September 2010.
　The capacity of bone tissue to alter its mass and architecture in response to mechanical request has long been known.　Bone not only develops as a structure designed specifically for mechanical demands, but it can adapt during life toward more efficient mechanical performance.　In particular, the skeletal effects of microgravity result in the development of an osteoporotic phenotype with several bone defects including a bone mass decrease resembling the bone modifications occurring in elder people and in bed rest conditions.　This is particularly true for weight bearing bones such as spine, femur and tibiae.　In contrast non-weight bearing bones like calvaria etc. didn't show bone mineral density decrease in weightlessness.　Given the interest of our laboratory in the microgravity induced skeleton alterations, we focused our attention on a transgenic mouse over-expressing pleiotrophin （PTN） under the control of the bone specific human osteocalcin promoter.　This protein is a heparin-binding cytokine with different functions.　In particular PTN-transgenic mice （PTN-Tg） show an increase in the bone mass and mineralization, with a calcium content/mg bone of 10.　We used this mouse model in the MDS flight experiment to study the PTN potential role in counteracting bone loss in microgravity.　In this transgenic line males have a higher bone mineral density and, compared to the wild type strain, bone loss is partially restored in ovariectomized females.　Three PTN-transgenic mice （Tg） and three wild type （Wt） mice were housed in the MDS （Mouse Drawer System） at the ISS for three months.　
　Unfortunately, during the three month period in space, three mice （two Wt and one Tg） died due to a spinal cord lesion probably occurred during the shuttle lift off, a possible liver pathology and a failure of the food delivery system respectively.　All the three dead mice were however frozen for subsequent skeletal analysis.　The remaining three mice had a normal behavior during the flight and appeared in excellent health conditions at the time of landing.　During the MDS stay at the ISS several physical parameters were under daily check.　With regard to the animal health status checking, the daily water consumption for each individual mouse revealed to be one of the most important parameter.　From blood cell analysis after landing, no major hematological alterations were noticed in the blood cell count except a slight increase in the number of erythrocytes.　The serum collected from these mice is being used in a Luminex panel assay for several cytokine and bone metabolism markers.　Immediately after return to earth, the mice were sacrificed, blood parameter were measured, all different tissues were dissected and the different bones isolated.　
　To study the microgravity effects on both wt and PTN-Tg mice we performed a microarchitectural study by synchrotron and bench microCT both at the Grenoble and the Trieste facilities.　With this last technique we analyzed both weight and non-weight bearing bones and we evaluated bone mineral density, mineralization amount, trabecular architecture.　We are also in the process of obtaining a holotomographic reconstruction of the trabecular and cortical bone from both the flight and the control mice.　In addition we extracted RNA from long bones and bone marrow of the same mice and we performed Real-time PCR analysis to determine the expression of bone marker such as osteocalcin, runx2, bone sialoprotein and of markers of bone turnover such as RankL, TRAP, cathepsin K, IL6 in the different animals.
　In order to obtain from the animals sent to the ISS as much as possible information including also microgravity induced modifications of tissues other than bone, we associated to the MDS experiment several international group from Italian, American, Japanese Universities and from NASA and JAXA labs and we created a Tissue Sharing Program （TSP）.　In total 17 groups from 6 countries were involved in the program.　Samples from almost the entire organism are now under investigation by the TSP team.　Initial results from these appear very promising.　For example, it was observed a modification of the thyroid structure and also the values of cAMP production after treatment with 10-7 M TSH for 1 hour were significantly lower than those obtained in Earth's gravity （FS. Ambesi et al. Udine, Italy）.　A characterization of the behavioral repertoire （ethogram） of the mice exposed to space environment is also under investigation based on the movie camera records.