Transcriptional Pathways Associated with Skeletal Muscle Changes after Spinal Cord Injury and Treadmill Locomotor Training.

The genetic and molecular events associated with changes in muscle mass and function after SCI and after the implementation of candidate therapeutic approaches are still not completely known. The overall objective of this study was to identify key molecular pathways activated with muscle remodeling after SCI and locomotor training. We implemented treadmill training in a well-characterized rat model of moderate SCI and performed genome wide expression profiling on soleus muscles at multiple time points: 3, 8, and 14 days after SCI. We found that the activity of the protein ubiquitination and mitochondrial function related pathways was altered with SCI and corrected with treadmill training. The BMP pathway was differentially activated with early treadmill training as shown by Ingenuity Pathway Analysis. The expression of several muscle mass regulators was modulated by treadmill training, including Fst, Jun, Bmpr2, Actr2b, and Smad3. In addition, key players in fatty acids metabolism (Lpl and Fabp3) responded to both SCI induced inactivity and reloading with training. The decrease in Smad3 and Fst early after the initiation of treadmill training was confirmed by RT-PCR. Our data suggest that TGFβ/Smad3 signaling may be mainly involved in the decrease in muscle mass observed with SCI, while the BMP pathway was activated with treadmill training

NASA's Rodent Research Project: Validation of Capabilities for Conducting Long Duration Experiments in Space

Research using rodents is an essential tool for advancing biomedical research on Earth and in space. Prior rodent experiments on the Shuttle were limited by the short flight duration. The International Space Station (ISS) provides a new platform for conducting rodent experiments under long duration conditions. Rodent Research (RR)-1 was conducted to validate flight hardware, operations, and science capabilities that were developed at the NASA Ames Research Center. Twenty C57BL6J adult female mice were launched on Sept 21, 2014 in a Dragon Capsule (SpaceX-4), then transferred to the ISS for a total time of 21-22 days (10 commercial mice) or 37 days (10 validation mice). Tissues collected on-orbit were either rapidly frozen or preserved in RNAlater at -80C (n2group) until their return to Earth. Remaining carcasses on-orbit were rapidly frozen for dissection post-flight. The three controls groups at Kennedy Space Center consisted of: Basal mice euthanized at the time of launch, Vivarium controls housed in standard cages, and Ground Controls (GC) housed in flight hardware within an environmental chamber. Upon return to Earth, there were no differences in body weights between Flight (FLT) and GC at the end of the 37 days in space. Liver enzyme activity levels of FLT mice and all control mice were similar in magnitude to those of the samples that were processed under optimal conditions in the laboratory. Liver samples dissected on-orbit yielded high quality RNA (RIN8.99+-0.59, n7). Liver samples dissected post-flight from the intact, frozen FLT carcasses yielded RIN of 7.27 +- 0.52 (n6). Additionally, wet weights of various tissues were measured. Adrenal glands and spleen showed no significant differences in FLT compared to GC although thymus and livers weights were significantly greater in FLT compared to GC. Over 3,000 tissue aliquots collected post-flight from the four groups of mice were deposited into the Ames Life Science Data Archives for future Biospecimen Sharing Program. Together, the RR validation flight successfully demonstrates the capability to support long-duration experimentation on the ISS to achieve both basic science and biomedical objectives

GeneLab: "Omics" Data Systems for Translational Space Biology Research

No abstract availabl

Preservation of Multiple Mammalian Tissues to Maximize Science Return from Ground Based and Spaceflight Experiments

<div><p>Background</p><p>Even with recent scientific advancements, challenges posed by limited resources and capabilities at the time of sample dissection continue to limit the collection of high quality tissues from experiments that can be conducted only infrequently and at high cost, such as in space. The resources and time it takes to harvest tissues post-euthanasia, and the methods and duration of long duration storage, potentially have negative impacts on sample quantity and quality, thereby limiting the scientific outcome that can be achieved.</p><p>Objectives</p><p>The goals of this study were to optimize methods for both sample recovery and science return from rodent experiments, with possible relevance to both ground based and spaceflight studies. The first objective was to determine the impacts of tissue harvest time post-euthanasia, preservation methods, and storage duration, focusing on RNA quality and enzyme activities in liver and spleen as indices of sample quality. The second objective was to develop methods that will maximize science return by dissecting multiple tissues after long duration storage <i>in situ</i> at -80°C.</p><p>Methods</p><p>Tissues of C57Bl/6J mice were dissected and preserved at various time points post-euthanasia and stored at -80°C for up to 11 months. In some experiments, tissues were recovered from frozen carcasses which had been stored at -80°C up to 7 months. RNA quantity and quality was assessed by measuring RNA Integrity Number (RIN) values using an Agilent Bioanalyzer. Additionally, the quality of tissues was assessed by measuring activities of hepatic enzymes (catalase, glutathione reductase and GAPDH).</p><p>Results</p><p>Fresh tissues were collected up to one hour post-euthanasia, and stored up to 11 months at -80°C, with minimal adverse effects on the RNA quality of either livers or RNAlater-preserved spleens. Liver enzyme activities were similar to those of positive controls, with no significant effect observed at any time point. Tissues dissected from frozen carcasses that had been stored for up to 7 months at -80°C had variable results, depending on the specific tissue analyzed. RNA quality of liver, heart, and kidneys were minimally affected after 6–7 months of storage at -80°C, whereas RNA degradation was evident in tissues such as small intestine, bone, and bone marrow when they were collected from the carcasses frozen for 2.5 months.</p><p>Conclusion</p><p>These results demonstrate that 1) the protocols developed for spaceflight experiments with on-orbit dissections support the retrieval of high quality samples for RNA expression and some protein analyses, despite delayed preservation post-euthanasia or prolonged storage, and 2) many additional tissues for gene expression analysis can be obtained by dissection even following prolonged storage of the tissue <i>in situ</i> at -80°C. These findings have relevance both to high value, ground-based experiments when sample collection capability is severely constrained, and to spaceflight experiments that entail on-orbit sample recovery by astronauts.</p></div

Summary of RIN values from harvested spleen and liver samples.

<p>Summary of RIN values from harvested spleen and liver samples.</p

Integrity of RNA harvested from whole frozen carcasses.

<p>Whole frozen carcasses (WC) were frozen using an aluminum block that was pre-chilled with liquid nitrogen to simulate the freezing kit used on-orbit (2.5, 4 and 6–7 month WC) prior to being placed into a -80°C freezer. Spleen, liver, heart, kidney, and lung were collected within 30 minutes post thawing of the WC, and preserved in RNAlater prior to analysis. Data sets were assessed for normality using the Shapiro-Wilk test, followed by the one-way ANOVA and Tukey’s post hoc test. Values are means ± SD (n = 9 for 2.5 month, except n = 8 for lung; n = 4, 5, 6, 3, 4 for 4 month spleen, liver, heart, kidney and lung, respectively; n = 3 for 6–7 month). * p<0.05; ** p<0.01</p

The effects on RNA quality in various tissues recovered from whole and partially dissected carcasses stored at -80°C for 2.5 months.

<p>Tissue samples were removed from the frozen carcasses for RIN analysis. Tissues were preserved in RNAlater prior to analysis. Brain, adrenal glands, eye, thymus, and hindlimb muscles resulted in high RIN values in both WC (A) and PC (B). RNA quality in WC and PC small intestine and bone marrow samples were severely degraded, resulting in RIN values < 3 (A and B). Values are means ± SD (n = 8 for Fig 7A, except n = 7 adrenal gland, n = 5 femur and bone marrow, and n = 4 tibia; n = 4 for Fig 7B, except n = 3 adrenal gland).</p