We would like to thank Alison French at NASA Ames Life Science Data Archive for her assistance with obtaining the video related to the Rodent Habitats and overall help with obtaining cage related information. We also like to thank Marla Smithwick at NASA Ames Research Center for her help with obtaining the proper information. Research funding was provided by the GeneLab Project at NASA Ames Research Center, through NASA's Space Biology Program in the Division of Space Life and Physical Sciences Research and Applications (SLPSRA). Any use of trade names is for descriptive purposes only and does not imply endorsement by the US Government.

The authors have nothing to disclose.

The NASA GeneLab platform is a comprehensive omics database and analysis platform that will allow the scientific community to generate novel hypotheses related to space biology. Here we have presented a comprehensive procedure for rodent experiments from the beginning of spaceflight to the generation of novel hypothesis from analyzing data utilizing a publicly available space biology platform. In addition, we have also provided an extensive protocol on an unbiased systems biology analysis for identifying key genes driving the system being studied. We have used our recent study36 as an example of how this protocol is effectively utilized to generate a novel hypothesis for space biology. We hope that this helps investigators better understand how spaceflight experiments are performed and how data from them lead to the data available on GeneLab, and ultimately allow for clearer interpretation of publicly available space biology omics data.
There are several critical steps within our protocol regarding both rodent spaceflight experiments and analysis of the data produced. Understanding the Rodent Habitat setup is critical to develop and design the optimal experiment for spaceflight. This would specifically entail the protocol and description we have provided in step 1 of our protocol. Once an investigator fully understands the different conditions existing in the Rodent Habitats compared to vivarium cages, the biological results being interpreted can be correlated properly to the environmental conditions in space. In additions, modifications to the Rodent Habitat cannot be done, since the Rodent Habitat has been optimally designed and approved by NASA for use of spaceflight.
To interpret the biological results, we have provided a thorough protocol on every step involved from uploading your data to GeneLab to analysis of the data to generate novel space biology hypothesis. Although all the steps are important in understanding how to generate data, the most critical steps for data analysis are steps 9 and 10. Step 9 provides a protocol to analyze transcriptomic data using an unbiased systems biology method to determine genes/pathways that are truly driving the experimental condition being analyzed. Step 10 is critical as it provides users with an easy methodology to analyze omics GeneLab datasets using the GeneLab platform. Modifications to the protocol provided can be done for some steps regarding analyzing data. Specifically, steps 9.4&#8211;9.6 can be done using R programming or any other favorite tools that the user prefers. Depending on the dataset, different statistics and fold-change cutoffs can be used to determine the significantly regulated genes. In addition, for determining the key genes in steps 9.5 and 9.6, the user can modify this protocol and use any tool that utilizes the significantly regulated genes to predict functions. The important concept is that using multiple predictive functional omics tools allows for determination of genes involved with the majority of functions being regulated in the system being studied.
The GeneLab platform continues to develop, and while the analyses described here were performed after data download, the next phase of GeneLab will allow for analysis of omics data directly on GeneLab platform, which will provide an easy workflow to generate processed data for higher-order analysis. Moreover, whereas we have focused on a protocol for interpreting transcriptomic data, GeneLab contains a wide variety of omics data including proteomic, genomic, metabolomic, and epigenomic data. The eventual platform will contain pipelines and guidelines for analysis of these different types of omics. The last phase of GeneLab will also implement a system level visualization interface to allow the basic user to easily generate space biology hypotheses.
Lastly, our systems biology analysis provides a unique and unbiased method to determine the key driving genes/pathways in any system being studied using omics datasets. We have used this methodology in several different independent studies with great success to determine the key drivers involved36,45,46,47,48,49,50. In a cancer related omics study, using this methodology we experimentally validated that our predicted key genes/pathways were actually driving the drug treatment response by knocking out the key genes in vitro45. We observed, as we had predicted through this protocol, that the treatment was not effective anymore due to the absence of the key genes. We believe that this unbiased systems biology protocol can be a useful tool to determine key pathways for any omics study.
This protocol provides a quick and efficient method for the generation of novel space biology hypotheses. The data generated from GeneLab can be leveraged by investigators for future funding opportunities, experimental validation, and potential targets for development of countermeasures against microgravity and space radiation. The protocol provided here will permit for future space biology investigations to occur with optimal efficiency to allow for safe long-term space missions.

The overall goal of this manuscript is to provide a clear methodology of how to use NASA&#39;s GeneLab platform1 and how rodent experiments done in space are translated to omics data for analysis. Spacefaring humans are exposed to numerous health risks from altered gravity fields, space radiation, isolation from Earth, and other hostile environmental factors2,3,4,5,6. Biological experiments performed in space and on the ground have helped to define and quantify these risks7,8,9,10,11,12,13,14. In space, these experiments have been conducted on the International Space Station (ISS), the Space Shuttle, and other orbital platforms. Conducting these experiments requires specialized hardware and methodology given the unique concerns of performing experiments in space including limited crew time and the microgravity environment. Various platforms now exist for performing sophisticated experiments in space using plant, animal, and microbial models15.
Rodent models have been particularly important to advancing our understanding of how mammals, including humans, respond to spaceflight. These include the impact of spaceflight on the muscle structure16,17,18 and immune functions19,20,21. The standard vivarium cages used for housing rodents on Earth are not suitable for spaceflight experiments22,23. Therefore, over the years mice and rats have been flown and housed in various cages including the Japanese Aerospace Exploration Agency (JAXA) Habitat Cage24, animal carrying space capsules used on the BION-M1 unmanned Russian satellite25,26,27, the Mice Drawer System (MDS) designed by the Italian Space Agency28,29,30, the NASA Animal Enclosure Module (AEM), and now the NASA Rodent Transporter and Habitats23. Rodent experiments first started on board the Space Shuttle using cages referred to as the Animal Enclosure Module (AEM). This hardware was used in 27 rodent experiments on the Space Shuttle23. The AEM was originally developed for relatively short experiments on-board the shuttle (&#60; 20 days). Since the development of the ISS, the AEMs have been modified for longer duration experiments and are now referred to as Rodent Habitats22,23. The new Rodent Habitats are designed to support long-duration missions in the ISS using the EXpedite the PRocessing of Experiments for Space Station (EXPRESS) Rack interface. Rodent Habitats are substantially different from standard vivarium cages in their dimensions, air flow, filter and exhaust system, and access to food and water (Figure 1). Nevertheless, this hardware has proven to be an effective research platform, enabling key insights into the spaceflight-induced changes to mammalian physiology19,31,32,33,34,35,36.
Large volumes of omics data can now be generated from biological spaceflight experiments including those performed with rodents. Recently, data from these omics experiments have been made publicly available through the NASA GeneLab platform1 which is a comprehensive data repository and analysis platform that allows anyone to develop hypotheses from spaceflight experiments. GeneLab provides tools for the discovery, access, sharing and analysis of data. We utilized GeneLab datasets to show that differences between the standard vivarium cages and specialized Rodent Habitats used in space cause massive differences in the transcriptome of mice36. We analyzed four different publicly available datasets, comparing different tissues from rodents housed in either the Rodent Habitats or standard vivarium cages. Using an unbiased systems biology analysis, we determined that the main drivers and pathways that were changed were consistent with a hypoxic response due to the high CO2 levels caused by higher CO2 concentrations on ISS, which leads to higher CO2 concentrations in the Rodent Habitat given that they are passive systems that take in the ambient air. This demonstrates how scientists can use open source tools and data to generate novel findings with implication on how the environment of the ISS impacts astronaut health.
Here we describe how rodent experiments are performed in space and how data from these experiments can be accessed through an open-source, omic platform related to space biology. We discuss the configuration of the Rodent Habitats used for space missions, and how spaceflight tissues are processed. We also describe how spaceflight omics data can be discovered and accessed on GeneLab and how key factors driving the overall response to spaceflight can be identified36. The specific example we will present on how this protocol is implemented will be comparing the biological differences occurring in rodents housed in Rodent Habitat and the vivarium controls that were published by Beheshti et al.36. It is important to note that ground controls are essential for spaceflight rodent experiments. As described in this protocol, these controls are done with both identical conditions (i.e., CO2 conditions, humidity, temperature, cage dimensions, etc.) in the Rodent Habitats on the ISS and in standard vivarium cages that have the standard environmental (i.e., CO2 conditions, humidity, and temperature) conditions on Earth. The rodents housed in the Rodent Habitat ground controls allow for the direct comparison to rodents in space. While rodents housed in vivarium cages allow for the biological comparison between the different housing (e.g., vivarium cages vs. Rodent hardware). The Rodent Habitat is different than vivarium cages in that it has constant air flow (0.1&#8211;0.3 m/s), a long duration, and a secondary exhaust filter that captures and absorbs the animal waste guided to the exhaust filter by continuous air flow in microgravity. In addition, Rodent Habitats have passive systems and intake ambient air; therefore, they also have higher CO2 concentrations due to elevated levels in the ISS cabin (~5,000 ppm).

<table><tbody><tr><td>C57BL/6 Mice</td><td>The Jackson Laboratoy</td><td>C57BL/6J</td><td>C57BL/6 mice were used for datasets related to Rodent Research-1 experiments</td></tr><tr><td>BALB/C Mice</td><td>Taconic</td><td>BALB</td><td>BALB/C mice were used for datasets related to Rodent Research-3 experiments</td></tr><tr><td>Vivarium Cages</td><td>Charles River Laboratory</td><td></td><td>Standard murine cages purchased from Charles River Laboratory</td></tr><tr><td>Rodent Habitat</td><td>NASA</td><td></td><td>This cage and all components are built internally at NASA</td></tr><tr><td>RNAlater</td><td>ThermoFisher Scientific</td><td>AM7020</td><td>RNAlater is used to store the tissue for further RNA isolation</td></tr></tbody></table>

exploring the effects of spaceflight on mouse physiology using the open access nasa genelab platform

Performing biological experiments in space requires special accommodations and procedures to ensure that these investigations are performed effectively and efficiently. Moreover, given the infrequency of these experiments it is imperative that their impacts be maximized. The rapid advancement of omics technologies offers an opportunity to dramatically increase the volume of data produced from precious spaceflight specimens. To capitalize on this, NASA has developed the GeneLab platform to provide unrestricted access to spaceflight omics data and encourage its widespread analysis. Rodents (both rats and mice) are common model organisms used by scientists to investigate space-related biological impacts. The enclosure that house rodents during spaceflight are called Rodent Habitats (formerly Animal Enclosure Modules), and are substantially different from standard vivarium cages in their dimensions, air flow, and access to water and food. In addition, due to environmental and atmospheric conditions on the International Space Station (ISS), animals are exposed to a higher CO2 concentration. We recently reported that mice in the Rodent Habitats experience large changes in their transcriptome irrespective of whether animals were on the ground or in space. Furthermore, these changes were consistent with a hypoxic response, potentially driven by higher CO2 concentrations. Here we describe how a typical rodent experiment is performed in space, how omics data from these experiments can be accessed through the GeneLab platform, and how to identify key factors in this data. Using this process, any individual can make critical discoveries that could change the design of future space missions and activities.

Determining key drivers from spaceflight transcriptomic data will assist NASA with determining health risks and developing potential countermeasures to combat negative effects on astronaut health. In our recent publication, we have followed the steps above and utilized GeneLab datasets to successfully show a novel finding that CO2 concentrations on the ISS can impact health36. We have also used the technique above in other studies to successfully determine the key factors driving the system being studied45,46,47,48,49,50. Here we will show how the results from using this protocol can be successfully used to determine the key drivers.
In this study, we primarily focused on the biological differences that occur in rodents housed in the Rodent Habits ground controls and the vivarium controls. As described above, it is the key to better understanding these two habitats, which will provide us information on possible confounding factors that can impact health due to the environment on the ISS. For all rodent spaceflight experiments, these ground controls are also essential to determine which biological factors are associated directly with spaceflight or due to the environmental conditions on the ISS. As stated in the protocol, the environmental condition for the vivarium habitat is not exposed to the higher CO2 level that is present for the Rodent Habitat. The vivarium habitat has the normal CO2 level that is present on Earth (currently being 300 to 380 ppm). The temperature and humidity for both habitats are similar.
We used the following datasets from the GeneLab platform to determine the key genes between the rodents housed in the Rodent Habitat ground controls and vivarium ground controls that are responsible for driving the differences between the two habitats: GLDS-21, GLDS-111, GLDS-25, and GLDS-63. Analysis to determine the significant genes was carried out as described above between the Rodent Habitat (previously AEM) and vivarium controls independently for each dataset. PCA plots showed grouping of the biological replicates (Figure 4 shows the PCA plots for GLDS-21). From the pre-processed data, we determined the leading-edge genes from the different GSEA gene sets. Using the genes with 1.2-fold-change (log2), we were able to predict the genes involved with predictions for upstream regulators, canonical pathways, and biofunctions. For each dataset we then found the common/overlapping genes involved for all the genes (Figure 5). These genes are now believed to be driving the response between the rodents in the Rodent Habitats (or AEM) and vivarium controls. Network representation of how these key genes connect shows the central hubs for each dataset being analyzed (Figure 6). For example, MAPK1 is the central hub for STS-108 skeletal muscle tissues from mice (Figure 6A). This would be interpreted as the gene that is driving the key genes and most likely the central player for causing biological differences for mice housed in Rodent Habitats versus the vivarium cages. In our previous work, we discuss how these key genes are associated with CO2 response from the existing scientific literature and how these genes can be responsible for biological changes observed in the mice36.
Taking a systems biology approach, we next determined a &#34;master regulator&#34; that connects all the datasets/tissues and is potentially responsible for universal biological effects in rodents housed in AEMs compared to vivarium cages. This was done by determining the gene from all the datasets that is the most connected when constructing a network from all the key genes. We were able to show that MAPK1 is the most connected gene and central hub from all the key genes (Figure 7). To confirm if MAPK1 might be responsible for biological changes in mice from the higher CO2 levels in AEMs, we looked through the scientific literature for supporting evidence. We found several studies indicating the correlation of MAPK1 with CO259 and hypoxia19,60,61.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58447/58447fig1.jpg" /> 
Figure 1: The Rodent Habitat (previously AEM) compared to the vivarium cages.&#160;(A) Image of the AEM cage provided by NASA (Credits: NASA/Dominic Hart). (B) The standard vivarium cage that is currently used (photo taken by our laboratory). This figure has been modified from Beheshti et al.36. <a href="https://www.jove.com/files/ftp_upload/58447/58447fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58447/58447fig2.jpg" /> 
Figure 2: The Rodent Habitat Hardware System with the three different modules involved during transportation to and from the space missions. The left module (A) is the Rodent Habitat module (previously AEM), the center module (B) is the Transporter, and the right module (C) is the Animal Access Unit (AAU). (D)The&#160;Mouse Transfer Box (MTB). (Credits: NASA/Dominic Hart). <a href="https://www.jove.com/files/ftp_upload/58447/58447fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58447/58447fig3.jpg" /> 
Figure 3:&#160;Example analysis workflow which can be used in the GeneLab Galaxy interface to process RNA-seq data. <a href="https://www.jove.com/files/ftp_upload/58447/58447fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58447/58447fig4.jpg" /> 
Figure 4: Principal component analysis (PCA) of representative dataset after pre-processing steps. GLDS-21 dataset for AEM vs. vivarium cage is shown for the murine skeletal muscle from the STS-118 mission. <a href="https://www.jove.com/files/ftp_upload/58447/58447fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58447/58447fig5.jpg" /> 
Figure 5:&#160;Venn diagram representing what key genes are determined using different pathway prediction tools. <a href="https://www.jove.com/files/ftp_upload/58447/58447fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58447/58447fig6.jpg" /> 
Figure 6: The key genes determined for all conditions and murine tissues between the AEM vs. vivarium cages.&#160;(A-E) Network representation of the key genes for each dataset/rodent tissue. Log2 fold-changes (with a cutoff of 1.2-fold-change) to the gene expression were used to obtain different shades of green for fold-change in downregulated genes, while different shades of red depict fold-change in upregulated genes. The darker the shade of green or red, the greater the fold-change. This figure has been modified from Beheshti et al.36. <a href="https://www.jove.com/files/ftp_upload/58447/58447fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58447/58447fig7.jpg" /> 
Figure 7: Determining the &#34;master regulator&#34; for rodents in Rodent Habitat housing compared to vivarium cages. Connections between all individual key genes (Figure 6) were determined and displayed as a network through IPA. Network is represented as a radial plot with the most connected key gene, MAPK1, in the center. <a href="https://www.jove.com/files/ftp_upload/58447/58447fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure 1: GeneLab-GenomeSpace Integration with ISACreator for Streamlining Data Processing Operations.&#160;&#160;<a href="https://www.jove.com/files/ftp_upload/58447/Supplemental_Figure_1.tif" target="_blank">Please click here to download this figure.</a>
Supplemental Figure 2: Screenshot of GeneLab searches using federation/integration with heterogeneous bioinformatics external databases (GEO, PRIDE, MG-RAST).&#160;<a href="https://www.jove.com/files/ftp_upload/58447/Supplemental_Figure_2.tif" target="_blank">Please click here to download this figure</a>
Supplemental Figure 3: Screenshot of the GeneLab collaborative workspace showing the user account management, and access controls (e.g., private, shared, public folders).&#160;<a href="https://www.jove.com/files/ftp_upload/58447/Supplemental_Figure_3.tif" target="_blank">Please click here to download this figure</a>

Watch this Scientific Journal Video about Exploring the Effects of Spaceflight on Mouse Physiology using the Open Access NASA GeneLab Platform at JoVE.com

Exploring the Effects of Spaceflight on Mouse Physiology using the Open Access NASA GeneLab Platform

The NASA GeneLab platform provides unfettered access to precious omics data from biological spaceflight experiments. We describe how a typical mouse experiment is conducted in space and how data from such experiments can be accessed and analyzed.

Performing biological experiments in space requires special accommodations and procedures to ensure that these investigations are performed effectively ...

exploring-effects-spaceflight-on-mouse-physiology-using-open-access

Research

JoVE Journal

Genetics

12.2K Views.  NASA Ames Research Center. The NASA GeneLab platform provides unfettered access to precious omics data from biological spaceflight experiments. We describe how a typical mouse experiment is conducted in space and how data from such experiments can be accessed and analyzed.

Video: Exploring the Effects of Spaceflight on Mouse Physiology using the Open Access NASA GeneLab Platform

Mouse Short- and Long-term Locomotor Activity Analyzed by Video Tracking Software

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice and other rodents. Improvements in video tracking software (VTS) have allowed it to be coupled to LMA testing, dramatically improving specificity and sensitivity when compared to the line crossings method with manual scoring. In addition, VTS enables high-throughput experimentation. While similar to automated video tracking used for the open field test (OFT), LMA testing is unique in that it allows mice to remain in their home cage and does not utilize the anxiogenic stimulus of bright lighting during the active phase of the light-dark cycle. Traditionally, LMA has been used for short periods of time (mins), while longer movement studies (hrs-days) have often used implanted transmitters and biotelemetry. With the option of real-time tracking, long-, like short-term LMA testing, can now be conducted using videography. Long-term LMA testing requires a specialized, but easily constructed, cage so that food and water (which is usually positioned on the cage top) does not obstruct videography. Importantly, videography and VTS allows for the quantification of parameters, such as path of mouse movement, that are difficult or unfeasible to measure with line crossing and/or biotelemetry. In sum, LMA testing coupled to VTS affords a more complete description of mouse movement and the ability to examine locomotion over an extended period of time.

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice. Coupling of video tracking software (VTS) and LMA allows for the improvement of specificity and sensitivity, especially when compared with the manual, line crossing method of LMA analysis. Additionally VTS allows long-term tracking of mouse LMA.

Locomotor  activity (LMA) is a simple and easily performed measurement of behavior in mice  and other rodents. Improvements in video tracking software ...

Behavior

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Cancer Research

Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the &#34;cloud&#34; and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in&#160;cabinet; two such cabinets (16 environments) may be controlled by one computer.

Fully automated system for measuring physiologically meaningful properties of the mechanisms mediating spatial localization, temporal localization, duration, rate&#160;and probability estimation, risk assessment, impulsivity, and the accuracy and precision of memory, in order to assess the effects of genetic and pharmacological manipulations on foundational mechanisms of cognition in mice.

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and ...

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of current interventions to adequately treat fatigue in all patients leaves a need for new treatment options. Despite the therapeutic need and importance of preclinical research in helping identify promising novel treatments, few preclinical assays of fatigue are available. Moreover, the most common preclinical assay used to assess fatigue-like behavior, voluntary wheel running, is not suitable for use with some strains of mice, may not be sensitive to drugs that reduce fatigue, and has relatively low throughput. The current protocol describes a novel, non-voluntary preclinical assay of fatigue-like behavior, the treadmill fatigue test, and provides evidence of its efficacy in detecting fatigue-like behavior in mice treated with a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in animals. This assay may be a beneficial alternative to wheel running, as fatigue-like behavior and potential interventions can be assessed in a greater number of mice over a shorter time frame, thus permitting faster discovery of new therapeutic options.

Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Developmental Biology

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal experimentation whenever possible. When there is no alternative to animal experimentation, the total number of laboratory animals used should be the minimum needed to obtain valuable data. Moreover, appropriate refinement measures should be applied to minimize pain, suffering, and distress accompanying the experimental procedure. The categories used to classify the degree of pain, suffering, and distress are non-recovery, mild, moderate, or severe (EU Directive 2010/63). To determine which categories apply in individual cases, it is crucial to use scientifically sound tools.
The well-being-assessment protocol presented here is designed for procedures during which general anesthesia is used. The protocol focuses on home cage activity, the Mouse Grimace Scale, and luxury behaviors such as burrowing and nest building behavior as indicators of well-being. It also uses the free exploratory paradigm for trait anxiety-related behavior. Fecal corticosterone metabolites as indicators of acute stress are measured over the 24-h post-anesthetic period.
The protocol provides scientifically solid information on the well-being of mice following general anesthesia. Due to its simplicity, the protocol can easily be adapted and integrated in a planned study. Although it does not provide a scale to classify distress in categories according to the EU Directive 2010/63, it can help researchers estimate the degree of severity of a procedure using scientifically sound data. It provides a way to improve the assessment of well-being in a scientific, animal-centered manner.

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered manner.

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

Mimicking a Space Mission to Mars Using Hindlimb Unloading and Partial Weight Bearing in Rats

Rodent ground-based models are widely used to understand the physiological consequences of space flight on the physiological system and have been routinely employed since 1979 and the development of hind limb unloading (HLU). However, the next steps in space exploration now include to travel to Mars where the gravity is 38% of Earth&#8217;s gravity. Since no human being has experienced this level of partial gravity, a sustainable ground-based model is necessary to investigate how the body, already impaired by the time spent in microgravity, would react to this partial load. Here, we used our innovative partial weight-bearing (PWB) model to mimic a short mission and stay on Mars to assess the physiological impairments in the hind limb muscles induced by two different levels of reduced gravity applied in sequential fashion. This could provide a safe, ground-based model to study the musculoskeletal adaptations to gravitational change and to establish effective countermeasures to preserve astronauts&#8217; health and function.

By using an innovative ground-based analogue model, we are able to simulate a space mission including a trip to (0 g) and a stay on Mars (0.38 g) in rats. This model allows for a longitudinal assessment of the physiological changes occurring during the two hypo-gravitational stages of the mission.

Rodent ground-based models are widely used to understand the physiological consequences of space flight on the physiological system and have been ...

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.

A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to ...

Medicine

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

Culturing Lymphocytes in Simulated Microgravity Using a Rotary Cell Culture System

Given the current limitations of conducting biological research in space, a few options exist for subjecting cell culture to simulated microgravity (SMG) on Earth. These options vary in their methods, principles, and suitability for use with suspension cell culture. Here, a cell culture method is described for subjecting lymphocytes to simulated microgravity using a commercially available rotary cell culture system, also known as a 2D clinostat or a rotating wall vessel (RWV) device. This cell culture method utilizes the principle of time-averaged gravity vector nullification to simulate microgravity by rotating the cells on a horizontal axis. The cells cultured in this system can be harvested and utilized in many different experimental assays to assess the effects of simulated microgravity on cellular function and physiology. The culturing technique may vary slightly depending on the cell type or line that is used, but the method described here may be applied to any suspension-type cell culture.

This is a step-by-step guide for using a commercially available rotary cell culture system to culture lymphocytes in simulated microgravity using specialized disposable culture vessels. This culturing method may be applied to any suspension-type cell culture.

Given the current limitations of conducting biological research in space, a few options exist for subjecting cell culture to simulated microgravity (SMG) ...

Exploring the Effects of Spaceflight on Mouse Physiology using the Open Access NASA GeneLab Platform

Summary

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Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

Mimicking a Space Mission to Mars Using Hindlimb Unloading and Partial Weight Bearing in Rats

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Culturing Lymphocytes in Simulated Microgravity Using a Rotary Cell Culture System