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Biospecimen Retrieval from NASA's Rodent Research-1: Maximizing Science Return from Flight Missions

Rodent Research (RR)-1 was conducted to validate flight hardware, operations, and science capabilities that were developed to support long duration missions on the International Space Station. After 37 days in microgravity twenty mice were euthanized and frozen on orbit. Upon return to Earth the carcasses were dissected and yielded 32 different types of tissues from each mouse and over 3200 tissue aliquots. Many tissues were distributed to the Space Life and Physical Sciences (SLPS) Biospecimen Sharing Program (BSP) Principal Investigators (PIs) through the Ames Life Science Data Archive (ALSDA). A second round of dissections was performed to collect additional tissues from the remaining carcasses thawed for a second time for additional BSP PIs. Tissues retrieved included vaginal walls, aorta, pelvis, brown adipose tissue, tail, spine and forearms. Although the analyses are still in progress, some of the PIs have reported that the quality of the tissues was acceptable for their study. In a separate experiment we tested the RNA quality of the tissues that were dissected from frozen carcasses that were subjected to euthanasia, freezing, first and second thaw dissections. Timelines simulated the on-orbit RR-1 procedures to assess the quality of the tissues retrieved from the second thaw dissections. We analyzed the RIN values of select tissues including kidney, brain, white adipose tissue (WAT) and brown adipose tissue (BAT). Overall the RIN values from the second thaw were lower compared to those from the first by about a half unit; however, the tissues yielded RNA that are acceptable quality for some quantitative gene expression assays. Interestingly, RIN values of brain tissues were 8.4+/-0.6 and 7.9+/-0.7 from first and second round dissections, respectively (n5). Kidney and WAT yielded RIN values less than 8 but they can still be used for qPCR. BAT yielded higher quality RNA (8.2+/-0.5) than WAT (5.2+/-20.9), possibly due to the high fat content. Together, these data show that select tissues can be utilized for gene expression studies even if they are retrieved from carcasses that were subjected to at least two freezing and thawing processes; this further expands science return from valuable and infrequent rodent experiments in space.

rodents↗

GeneLab: Multi-Omics Investigation of Rodent Research-1 Bio-Banked Tissues

NASAs Rodent Research (RR) project is playing a critical role in advancing biomedical research on the physiological effects of space environments. Due to the limited resources for conducting biological experiments aboard the International Space Station (ISS), it is imperative to use crew time efficiently while maximizing high-quality science return. NASAs GeneLab project has as its primary objectives to 1) further increase the value of these experiments using a multi-omics, systems biology-based approach, and 2) disseminate these data without restrictions to the scientific community. The current investigation assessed viability of RNA, DNA, and protein extracted from archived RR-1 tissue samples for epigenomic, transcriptomic, and proteomic assays. During the first RR spaceflight experiment, a variety of tissue types were harvested from subjects, snap-frozen or RNAlater-preserved, and then stored at least a year at -80OC after return to Earth. They were then prioritized for this investigation based on likelihood of significant scientific value for spaceflight research. All tissues were made available to GeneLab through the bio-specimen sharing program managed by the Ames Life Science Data Archive and included mouse adrenal glands, quadriceps, gastrocnemius, tibialis anterior, extensor digitorum longus, soleus, eye, and kidney. We report here protocols for and results of these tissue extractions, and thus, the feasibility and value of these kinds of omics analyses. In addition to providing additional opportunities for investigation of spaceflight effects on the mouse transcriptome and proteome in new kinds of tissues, our results may also be of value to program managers for the prioritization of ISS crew time for rodent research activities. Support from the NASA Space Life and Physical Sciences Division and the International Space Station Program is gratefully acknowledged.

GeneLab↗

Rodent Research in Space: A Decadal Research Campaign

Studying the physiological effects of spaceflight in rodents is imperative to our understanding of adaptation and to providing countermeasures. We posit that studies of the reproductive system, development, stem cells, and behavior/cognition are critically important and provide outstanding opportunities as these are sentinel tissues and systems for radiation exposure and overall health. To improve understanding of the environmental stressors (weightlessness, space radiation, isolation/confinement) that impact space travelers, rodent research should be continued within and extended beyond LEO.

rodent research spaceflight↗

Machine Learning Models to Predict Cognitive Impairment of Rodents Subjected to Space Radiation

This research uses machine-learned computational analyses to predict the cognitive performance impairment of rats induced by irradiation. The experimental data in the analyses is from a rodent model exposed to ≤ 15 cGy of individual Galactic Cosmic Radiation (GCR) ions: 4He, 16O, 28Si, 48Ti, or 56Fe, expected for a Lunar or Mars mission. This work investigates rats at a subject-based level and uses performance scores taken before irradiation to predict impairment in Attentional Set-shifting (ATSET) data post-irradiation. Here, the worst performing rats of the control group define the impairment thresholds based on population analyses via cumulative distribution functions, leading to the labeling of impairment for each subject. A significant finding is the exhibition of a dose-dependent increasing probability of impairment for 1 to 10 cGy of 28Si or 56Fe in the Simple Discrimination (SD) stage of the ATSET, and for 1 to 10 cGy of 56Fe in the Compound Discrimination (CD) stage. On a subject-based level, implementing Machine Learning (ML) classifiers such as the Gaussian Naïve Bayes, Support Vector Machine, and Artificial Neural Networks identifies rats that have a higher tendency for impairment after GCR exposure. The algorithms employ the experimental prescreenperformance scores as multidimensional input features to predict each rodent’s susceptibility to cognitive impairment due to space radiation exposure. The receiver operating characteristic and the precision-recall curves of the ML models show a better prediction of impairment when 56Feis the ion in question in both SD and CD stages. They, however, do not depict impairment due to 4Hein SD and 28Siin CD, suggesting no dose-dependent impairment response in these cases. One key finding of our study is that prescreen performance scores can be used to predict the ATSET performance impairments. This result is significant to crewed space missions as it supports the potential of predicting an astronaut’s impairment in a specific task before spaceflight through the implementation of appropriately trained ML tools. Future research can focus on constructing ML ensemble methods to integrate the findings from the methodologies implemented in this study for morerobust predictionsof cognitive decrements due to space radiation exposure.

space radiation↗

Rodent growth, behavior, and physiology resulting from flight on the Space Life Sciences-1 mission

A rodent-based spaceflight study is conducted to investigate physiological changes in rats vs humans and the effects of changes in the design of the Research Animal Holding Facility (RAHF) and the Animal Enclosure Module (AEM). Rats were housed in the AEM and the RAHF, and controls were kept in identical flight hardware on earth subjected to the same flight-environmental profile. Biosamples and organ weights are taken to compare the rats before and after flight, and food/water intake are also compared. Weight gain, body weight, and food consumptions in the flight rats are significantly lower than corresponding values for the control subjects. Flight rats tend to have smaller postexperiment spleens and hearts, and flight rats consumed more water in the AEM than in the RAHF. The rodents' behavior is analogous to humans with respect to physiological and reconditioning effects, showing that the rat is a good model for basic research into the effects of spaceflight on humans.

Jahns, G.↗

Effects of Cage Type and NASA Rodent Food Bar in Male Sprague-Dawley Rats

Early prototype caging for the rodent Advanced Animal Habitat (P-AAH) for the International Space Station (ISS) is currently being tested. In this five week study, effects of the wire-bottom P-AAH cages and specialized NASA rodent food bars (FB) were compared to standard vivarium cages (VIV) with corn-cob, litter-filled bottoms, and standard Purina rat chow (CH). Ninety-six male Sprague-Dawley rats were divided into four treatment groups (24 rats/treatment): Group 1) VIV+CH, Group 2) P-AAH+CH, Group 3) VIV+FB, and Group 4) P-AAH+FB. Each VIV and P-AAH cage housed three and six rats, respectively. After five weeks of treatment rats were weighed, euthanized, and blood samples were collected. Weights of liver (LIV), kidney (KID), brain (BRN), epididymal fat (EPI), and perirenal fat (PERI) were also measured. Statistical analysis to compare differences between groups was performed by standard analysis of variance procedures (ANOVA) with a significance level of pLO.05. Results indicated P-AAH housed rats had significantly lower body weights (BW), LIV weights, and LIV/BW than VIV housed rats. FB fed rats had significantly lower blood urea nitrogen (BUN) levels and LIV/BW than CH fed rats. In addition, FB fed rats had significantly higher cholesterol (CHOL) levels, EPI/BW, PERI/BW, and total fat (EPI+PERI)/BW than CH fed rats. The P-AAH+FB group had significantly lower EPI, BRN, and total fat than VIV+FB rats. VIV+FB rats had significantly higher BRN, EPI, PERI, and total fat than VIV+CH rats. Triglycerides (TG), KID, KID/BW, and BRN/BW were not significantly different among treatment groups. These findings provide valuable information regarding cage design and food bar suitability for long-term use on the ISS.

Lau, Angela↗

Rodent Biocompatibility Test Using the NASA Foodbar and Epoxy EP21LV

Epoxy has been used successfully to affix NASA foodbars to the inner walls of the Animal Enclosure Module for past space flight experiments utilizing rodents. The epoxy used on past missions was discontinued, making it necessary to identify a new epoxy for use on the STS-108 and STS-107 missions. This experiment was designed to test the basic biocompatibility of epoxy EP21LV with male rats (Sprague Dawley) and mice (Swiss Webster) when applied to NASA foodbars. For each species, the test was conducted with a control group fed untreated foodbars and an experimental group fed foodbars applied with EP21LV. For each species, there were no group differences in animal health and no statistical differences (P<0.05) in body weights throughout the study. In mice, there was a 16% increase in heart weight in the epoxy group; this result was not found in rats. For both species, there were no statistical differences found in other organ weights measured. In rats, blood glucose levels were 15% higher and both total protein and globulin were 10% lower in the epoxy group. Statistical differences in these parameters were not found in mice. For both species, no statistical differences were found in other blood parameters tested. Food consumption was not different in rats but water consumption was significantly decreased 10 to 15% in the epoxy group. The difference in water consumption is likely due to an increased water content of the epoxy-treated foodbars. Finally, both species avoided consumption of the epoxy material. Based on the global analysis of the results, the few parameters found to be statistically different do not appear to be a physiologically relevant effect of the epoxy material, We conclude that the EP21LV epoxy is biocompatible with rodents.

Tillman, J.↗

Pulmonary Toxicity Studies of Lunar Dusts in Rodents

NASA will build an outpost on the lunar surface for long-duration human habitation and research. The surface of the Moon is covered by a layer of fine, reactive dust, and the living quarters in the lunar outpost are expected to be contaminated by lunar dust. Because the toxicity of lunar dust is not known, NASA has tasked its toxicology laboratory to evaluate the risk of exposure to the dust and to establish safe exposure limits for astronauts working in the lunar habitat. Studies of the pulmonary toxicity of a dust are generally done first in rodents by intratracheal/intrapharyngeal instillation. This toxicity screening test is then followed by an inhalation study, which requires much more of the test dust and is labor intensive. Preliminary results obtained by examining lung lavage fluid from dust-treated mice show that lunar dust was somewhat toxic (more toxic than TiO2, but less than quartz dust). More extensive studies are in progress to further examine lung lavage fluid for biomarkers of toxicity and lung tissues for histopathological lesions in rodents exposed to aged and activated (ground) lunar dust samples. In these studies, reference dusts (TiO2 and quartz) of known toxicities and have industrial exposure limits will be studied in parallel so the relative toxicity of lunar dust can be determined. The results from the instillation studies will be useful for choosing exposure concentrations for the animal inhalation study. The animal inhalation exposure will be conducted with lunar dust simulant prior to the study with the lunar dust. The experiment with the simulate will ensure that the study techniques used with actual lunar dust will be successful. The results of instillation and inhalation studies will reveal the toxicological risk of exposures and are essential for setting exposure limits on lunar dust for astronauts living in the lunar habitat.

Lam, Chiu-wing↗

Maximizing Science Return from Future Rodent Experiments on the International Space Station (ISS): Tissue Preservation

To better understand how mammals adapt to long duration habitation in space, a system for performing rodent experiments on the ISS is under development; Rodent Research-1 is the first flight and will include validation of both on-orbit animal support and tissue preservation. To evaluate plans for on-orbit sample dissection and preservation, we simulated conditions for euthanasia, tissue dissection, and prolonged sample storage on the ISS, and we also developed methods for post-flight dissection and recovery of high quality RNA from multiple tissues following prolonged storage in situ for future science. Mouse livers and spleens were harvested under conditions that simulated nominal, on-orbit euthanasia and dissection operations including storage at -80 C for 4 months. The RNA recovered was of high quality (RNA Integrity Number, RIN(is) greater than 8) and quantity, and the liver enzyme contents and activities (catalase, glutathione reductase, GAPDH) were similar to positive controls, which were collected under standard laboratory conditions. We also assessed the impact of possible delayed on-orbit dissection scenarios (off-nominal) by dissecting and preserving the spleen (RNAlater) and liver (fast-freezing) at various time points post-euthanasia (from 5 min up to 105 min). The RNA recovered was of high quality (spleen, RIN (is) greater than 8; liver, RIN (is) greater than 6) and liver enzyme activities were similar to positive controls at all time points, although an apparent decline in select enzyme activities was evident at the latest time (105 min). Additionally, various tissues were harvested from either intact or partially dissected, frozen carcasses after storage for approximately 2 months; most of the tissues (brain, heart, kidney, eye, adrenal glands and muscle) were of acceptable RNA quality for science return, whereas some tissues (small intestine, bone marrow and bones) were not. These data demonstrate: 1) The protocols developed for future flight experiments will support science return despite delayed preservation post-euthanasia or prolonged storage, and 2) Many additional tissues for gene expression analysis can be obtained by dissection following prolonged storage of the tissue in situ at -80 C. These findings have relevance both to high value, ground-based experiments when sample collection capability is severely constrained, and to all future spaceflight experiments that entail on-orbit sample recovery by the ISS crew.

tissues↗

Evaluation of Rodent Spaceflight in the NASA Animal Enclosure Module for an Extended Operational Period (up to 35 days)

The National Aeronautics and Space Administration Animal Enclosure Module (AEM) was developed as a self-contained rodent habitat for shuttle flight missions that provides inhabitants with living space, food, water, ventilation, and lighting for shuttle flight missions, and this study reports whether, after minimal hardware modification, the AEM could support an extended term up to 35 days for Sprague-Dawley rats and C57BL/6 female mice for use on the International Space Station. Success was evaluated based on comparison of AEM housed animals to that of vivarium housed and to normal biological ranges through various measures of animal health and well-being, including animal health evaluations, animal growth and body masses, organ masses, rodent food bar consumption, water consumption, and analysis of blood contents. The results of this study confirmed that the AEMs could support 12 adult female C57BL/6 mice for up to 35 days with self-contained RFB and water, and the AEMs could also support 5 adult male Sprague-Dawley rats for 35 days with external replenishment of diet and water. This study has demonstrated the capability and flexibility of the AEM to operate for up to 35 days with minor hardware modification. Therefore, with modifications, it is possible to utilize this hardware on the International Space Station or other operational platforms to extend the space life science research use of mice and rats.

Animal enclosure module↗

Maximizing Science Return from Future Rodent Experiments on the International Space Station (ISS): Tissue Preservation

To better understand how mammals adapt to long duration habitation in space, a system for performing rodent experiments on the ISS is under development. Rodent Research-1 is the first flight and will include validation of both on-orbit animal support and tissue preservation. To evaluate plans for on-orbit sample dissection and preservation, we simulated conditions for euthanasia, tissue dissection, and prolonged sample storage on the ISS, and we also developed methods for post-flight dissection and recovery of high quality RNA from multiple tissues following prolonged storage in situ for future science return. Livers and spleens from mice were harvested under conditions that simulated nominal, on-orbit euthanasia and dissection procedures including storage at minus 80 degrees Centigrade for 4 months. The RNA recovered was of high quality (RNA Integrity Number, RNA Integrity Number (RIN) greater than 8) and quantity, and the liver enzyme contents and activities (catalase, glutathione reductase, GAPDH) were similar to positive controls, which were collected under standard laboratory conditions. We also assessed the impact of possible delayed on-orbit dissection scenarios (off-nominal) by dissecting and preserving the spleen (RNA, later) and liver (fast-freezing) at various time points post-euthanasia (from 5 minutes up to 105 minutes). The RNA recovered was of high quality (spleen, RIN greater than 8; liver, RIN greater than 6) and liver enzyme activities were similar to positive controls at all time points, although an apparent decline in select enzyme activities was evident at 105 minutes. Additionally, various tissues were harvested from either intact or partially dissected, frozen carcasses after storage for approximately 2 months; most of the tissues (brain, heart, kidney, eye, adrenal glands and muscle) were of acceptable RNA quality for science return, whereas some tissues (small intestine, bone marrow and bones) were not. These data demonstrate: 1) The protocols developed for future flight experiments will support science return despite delayed preservation post-euthanasia or prolonged storage, and 2) High-quality RNA samples from many different tissues can be recovered by dissection following prolonged storage of the tissue in situ at minus 80 degrees Centigrade. These findings have relevance both to high-value, ground-based experiments when sample collection capability is severely constrained, and to future spaceflight experiments that entail on-orbit sample recovery by the ISS crew.

tissues↗

Potential NASA Rodent Centrifuge

This presentation is a summary of the continuing effort to determine options for studying artificial gravity with rodents. Results of an engineering trade study are presented and an overview of past and planned short radius centrifugation studies are presented. A leading proposal for a future flight centrifuge capable of housing rodents, the Techshot RCF, is presented in only enough detail as is approved by Techshot for public domain use.

Donovan, Frances↗

Characterization of Sensory and Shelter Enrichment in the Rodent Research Habitat

The ISS provides a platform for conducting Rodent Research (RR) in microgravity and 9 missions have been successfully conducted. The results from these experiments have begun to provide new insights into the effects of spaceflight on mammalian physiological systems. After RR-1-4, the Flight IACUC required inclusion of additional cage enrichment into the Rodent Habitats (RH) to "enhance animal well-being by providing animals with sensory and motor stimulation, through structures and resources that facilitate the expression of species typical behaviors". A Hut, in the form of a rigid, mesh igloo-like shelter was implemented beginning with RR-5. The potential influence of the Hut in the novel cage environment of RH on various spaceflight-sensitive physiological systems has not been fully explored. To understand the effects of the Hut, mice (female C57Bl/6J, 15wks) were housed in Vivarium cage (n=5), RH with Hut (n=5), No Hut (n=5), Nestlet (n=10), and Cocoon (n=10) for 7 weeks. There were no differences in weekly body mass or food consumption. Tail blood draw indicated no differences in plasma corticosterone levels, immune cell types, or IgA levels. 24hrs prior to euthanasia, Open Field (OF) and Novel Object (NO) tests were performed. There were no differences across groups, all mice engaged in thigmotaxis (arena wall proximity) in the OF over 50% of the recorded time, and thigmotaxis declined when a NO was introduced. Additional behavioral analysis from daily videos are in progress to quantify activity levels. Post-euthanasia, there were no differences in soleus muscle or adrenal gland mass. Analysis of distal femur cancellous revealed some differences in microarchitecture. These results show that introduction of the Hut may diminish differences observed between spaceflight and ground controls, warranting improved validation of Hut effects in space, and also underscore the value of thorough preflight, ground based testing.

Shirazi-Fard, Yasaman↗

Rodent Research Capabilities Update

Space Biology current Rodent Research hardware and capabilities as of October 2017. These include the Life Sciences Glovebox, Rapid Freeze, sample cartridges and simulated carcass freezing, mass measurement device, habitat configuration with enrichment, and the non-Rodent capabilities they feature.

Leveson-Gower, Dennis↗

Environmental Enrichment in the ISS Rodent Habitat Hardware System

Responses of animals exposed to microgravity during in-space experiments were reviewed from NASAs and ESA available video recording archives. These documented observation of animal behavior, as well as the range and level of activities during spaceflight, clearly demonstrate that weightlessness conditions and the extreme novelty of the surroundings exert damaging psychological stresses on the inhabitants. In response to a recognized need for in-flight animals to improve their wellbeing we propose to reduce such stresses by shaping and interrelating structures and surroundings to satisfying vital physiological needs of inhabitants. Rodent Habitat Hardware System based housing facility incorporating a tubing network system, to maintain and monitor rodent health environment with advanced accessories has been proposed. The new tubing configuration was found suitable for further incorporation of innovative monitoring technology and accessories in the animal holding habitat unit which allow to monitor in real-time the most valuable health related biological parameter under weightlessness environment of spaceflight.

Orr, Sophie↗

OP3 NASA JAXA Rodent Research Collaboration Opportunities

Space Biology and Human Research Projects Integrated Proposal to use the JAXA MARS facility to be presented to JAXA and JAXA investigator audience at JAXA Kibo Utilization Symposium and OP3 negotiation for 2019 ISS rodent mission. Slides present a pictorial overview of the proposed science and analysis techniques desired from the US investigator team. Prior published data form a recent collaboration with JAXA is also mentioned.

JAXA Rodent Mouse Habitat Unit↗

Reduced volume of diabetic pancreatic islets in rodents detected by synchrotron X-ray phase-contrast microtomography and deep learning network

The pancreatic islet is a highly structured micro-organ that produces insulin in response to rising blood glucose. Here we develop a label-free and automatic imaging approach to visualize the islets in situ in diabetic rodents by the synchrotron radiation X-ray phase-contrast microtomography (SRμCT) at the ID17 station of the European Synchrotron Radiation Facility. The large-size images (3.2 mm × 15.97 mm) were acquired in the pancreas in STZ-treated mice and diabetic GK rats. Each pancreas was dissected by 3000 reconstructed images. The image datasets were further analysed by a self-developed deep learning method, AA-Net. All islets in the pancreas were segmented and visualized by the three-dimension (3D) reconstruction. After quantifying the volumes of the islets, we found that the number of larger islets (=>1500 μm 3 ) was reduced by 2-fold (wt 1004 ± 94 vs GK 419 ± 122, P < 0.001) in chronically developed diabetic GK rat, while in STZ-treated diabetic mouse the large islets were decreased by half (189 ± 33 vs 90 ± 29, P < 0.001) compared to the untreated mice. Our study provides a label-free tool for detecting and quantifying pancreatic islets in situ. It implies the possibility of monitoring the state of pancreatic islets in vivo diabetes without labelling.

59 BASIC BIOLOGICAL SCIENCES↗