Correction: Tran et al. Delayed Maturation of Oligodendrocyte Progenitors by Microgravity: Implications for Multiple Sclerosis and Space Flight. Life 2022, 12, 797.

The authors wish to make the following corrections to Figure 10 of this paper [...].

Delayed Maturation of Oligodendrocyte Progenitors by Microgravity: Implications for Multiple Sclerosis and Space Flight.

In previous studies, we examined the effects of space microgravity on human neural stem cells. To date, there are no studies on a different type of cell that is critical for myelination and electrical signals transmission, oligodendrocyte progenitors (OLPs). The purpose of the present study was to examine the behavior of space-flown OLPs (SPC-OLPs) as they were adapting to Earth's gravity. We found that SPC-OLPs survived, and most of them proliferated normally. Nonetheless, some of them displayed incomplete cytokinesis. Both morphological and ontogenetic analyses showed that they remained healthy and expressed the immature OLP markers Sox2, PDGFR-α, and transferrin (Tf) after space flight, which confirmed that SPC-OLPs displayed a more immature phenotype than their ground control (GC) counterparts. In contrast, GC OLPs expressed markers that usually appear later (GPDH, O4, and ferritin), indicating a delay in SPC-OLPs' development. These cells remained immature even after treatment with culture media designed to support oligodendrocyte (OL) maturation. The most remarkable and surprising finding was that the iron carrier glycoprotein Tf, previously described as an early marker for OLPs, was expressed ectopically in the nucleus of all SPC-OLPs. In contrast, their GC counterparts expressed it exclusively in the cytoplasm, as previously described. In addition, analysis of the secretome demonstrated that SPC-OLPs contained 3.5 times more Tf than that of GC cells, indicating that Tf is gravitationally regulated, opening two main fields of study to understand the upregulation of the Tf gene and secretion of the protein that keep OLPs at a progenitor stage rather than moving forward to more mature phenotypes. Alternatively, because Tf is an autocrine and paracrine factor in the central nervous system (CNS), in the absence of neurons, it accumulated in the secretome collected after space flight. We conclude that microgravity is becoming a novel platform to study why in some myelin disorders OLPs are present but do not mature.

Effects of spaceflight aboard the International Space Station on mouse estrous cycle and ovarian gene expression.

Ovarian steroids dramatically impact normal homeostatic and metabolic processes of most tissues within the body, including muscle, bone, neural, immune, cardiovascular, and reproductive systems. Determining the effects of spaceflight on the ovary and estrous cycle is, therefore, critical to our understanding of all spaceflight experiments using female mice. Adult female mice (n = 10) were exposed to and sacrificed on-orbit after 37 days of spaceflight in microgravity. Contemporary control (preflight baseline, vivarium, and habitat; n = 10/group) groups were maintained at the Kennedy Space Center, prior to sacrifice and similar tissue collection at the NASA Ames Research Center. Ovarian tissues were collected and processed for RNA and steroid analyses at initial carcass thaw. Vaginal wall tissue collected from twice frozen/thawed carcasses was fixed for estrous cycle stage determinations. The proportion of animals in each phase of the estrous cycle (i.e., proestrus, estrus, metestrus, and diestrus) did not appreciably differ between baseline, vivarium, and flight mice, while habitat control mice exhibited greater numbers in diestrus. Ovarian tissue steroid concentrations indicated no differences in estradiol across groups, while progesterone levels were lower (p < 0.05) in habitat and flight compared to baseline females. Genes involved in ovarian steroidogenic function were not differentially expressed across groups. As ovarian estrogen can dramatically impact multiple non-reproductive tissues, these data support vaginal wall estrous cycle classification of all female mice flown in space. Additionally, since females exposed to long-term spaceflight were observed at different estrous cycle stages, this indicates females are likely undergoing ovarian cyclicity and may yet be fertile.

Validation of a New Rodent Experimental System to Investigate Consequences of Long Duration Space Habitation.

Animal models are useful for exploring the health consequences of prolonged spaceflight. Capabilities were developed to perform experiments in low earth orbit with on-board sample recovery, thereby avoiding complications caused by return to Earth. For NASA's Rodent Research-1 mission, female mice (ten 32 wk C57BL/6NTac; ten 16 wk C57BL/6J) were launched on an unmanned vehicle, then resided on the International Space Station for 21/22d or 37d in microgravity. Mice were euthanized on-orbit, livers and spleens dissected, and remaining tissues frozen in situ for later analyses. Mice appeared healthy by daily video health checks and body, adrenal, and spleen weights of 37d-flight (FLT) mice did not differ from ground controls housed in flight hardware (GC), while thymus weights were 35% greater in FLT than GC. Mice exposed to 37d of spaceflight displayed elevated liver mass (33%) and select enzyme activities compared to GC, whereas 21/22d-FLT mice did not. FLT mice appeared more physically active than respective GC while soleus muscle showed expected atrophy. RNA and enzyme activity levels in tissues recovered on-orbit were of acceptable quality. Thus, this system establishes a new capability for conducting long-duration experiments in space, enables sample recovery on-orbit, and avoids triggering standard indices of chronic stress.

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.

Author Correction: Behavior of mice aboard the International Space Station.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Behavior of mice aboard the International Space Station.

Interest in space habitation has grown dramatically with planning underway for the first human transit to Mars. Despite a robust history of domestic and international spaceflight research, understanding behavioral adaptation to the space environment for extended durations is scant. Here we report the first detailed behavioral analysis of mice flown in the NASA Rodent Habitat on the International Space Station (ISS). Following 4-day transit from Earth to ISS, video images were acquired on orbit from 16- and 32-week-old female mice. Spaceflown mice engaged in a full range of species-typical behaviors. Physical activity was greater in younger flight mice as compared to identically-housed ground controls, and followed the circadian cycle. Within 7-10 days after launch, younger (but not older), mice began to exhibit distinctive circling or 'race-tracking' behavior that evolved into coordinated group activity. Organized group circling behavior unique to spaceflight may represent stereotyped motor behavior, rewarding effects of physical exercise, or vestibular sensation produced via self-motion. Affording mice the opportunity to grab and run in the RH resembles physical activities that the crew participate in routinely. Our approach yields a useful analog for better understanding human responses to spaceflight, providing the opportunity to assess how physical movement influences responses to microgravity.

Forces associated with launch into space do not impact bone fracture healing.

Segmental bone defects (SBDs) secondary to trauma invariably result in a prolonged recovery with an extended period of limited weight bearing on the affected limb. Soldiers sustaining blast injuries and civilians sustaining high energy trauma typify such a clinical scenario. These patients frequently sustain composite injuries with SBDs in concert with extensive soft tissue damage. For soft tissue injury resolution and skeletal reconstruction a patient may experience limited weight bearing for upwards of 6 months. Many small animal investigations have evaluated interventions for SBDs. While providing foundational information regarding the treatment of bone defects, these models do not simulate limited weight bearing conditions after injury. For example, mice ambulate immediately following anesthetic recovery, and in most cases are normally ambulating within 1-3 days post-surgery. Thus, investigations that combine disuse with bone healing may better test novel bone healing strategies. To remove weight bearing, we have designed a SBD rodent healing study in microgravity (µG) on the International Space Station (ISS) for the Rodent Research-4 (RR-4) Mission, which launched February 19, 2017 on SpaceX CRS-10 (Commercial Resupply Services). In preparation for this mission, we conducted an end-to-end mission simulation consisting of surgical infliction of SBD followed by launch simulation and hindlimb unloading (HLU) studies. In brief, a 2 mm defect was created in the femur of 10 week-old C57BL6/J male mice (n = 9-10/group). Three days after surgery, 6 groups of mice were treated as follows: 1) Vivarium Control (maintained continuously in standard cages); 2) Launch Negative Control (placed in the same spaceflight-like hardware as the Launch Positive Control group but were not subjected to launch simulation conditions); 3) Launch Positive Control (placed in spaceflight-like hardware and also subjected to vibration followed by centrifugation); 4) Launch Positive Experimental (identical to Launch Positive Control group, but placed in qualified spaceflight hardware); 5) Hindlimb Unloaded (HLU, were subjected to HLU immediately after launch simulation tests to simulate unloading in spaceflight); and 6) HLU Control (single housed in identical HLU cages but not suspended). Mice were euthanized 28 days after launch simulation and bone healing was examined via micro-Computed Tomography (µCT). These studies demonstrated that the mice post-surgery can tolerate launch conditions. Additionally, forces and vibrations associated with launch did not impact bone healing (p = .3). However, HLU resulted in a 52.5% reduction in total callus volume compared to HLU Controls (p = .0003). Taken together, these findings suggest that mice having a femoral SBD surgery tolerated the vibration and hypergravity associated with launch, and that launch simulation itself did not impact bone healing, but that the prolonged lack of weight bearing associated with HLU did impair bone healing. Based on these findings, we proceeded with testing the efficacy of FDA approved and novel SBD therapies using the unique spaceflight environment as a novel unloading model on SpaceX CRS-10.

Endothelial lipase is a major determinant of HDL level.

A new member of the lipase gene family, initially termed endothelial lipase (gene nomenclature, LIPG; protein, EL), is expressed in a variety of different tissues, suggesting a general role in lipid metabolism. To assess the hypothesis that EL plays a physiological role in lipoprotein metabolism in vivo, we have used gene targeting of the native murine locus and transgenic introduction of the human LIPG locus in mice to modulate the level of EL expression. Evaluation of these alleles in a C57Bl/6 background revealed an inverse relationship between HDL cholesterol level and EL expression. Fasting plasma HDL cholesterol was increased by 57% in LIPG(-/-) mice and 25% in LIPG(+/-) mice and was decreased by 19% in LIPG transgenic mice as compared with syngeneic controls. Detailed analysis of lipoprotein particle composition indicated that this increase was due primarily to an increased number of HDL particles. Phospholipase assays indicated that EL is a primary contributor to phospholipase activity in mouse. These data indicate that expression levels of this novel lipase have a significant effect on lipoprotein metabolism.

Payload hardware and experimental protocol development to enable future testing of the effect of space microgravity on the resistance to gentamicin of uropathogenic Escherichia coli and its σs-deficient mutant.

Human immune response is compromised and bacteria can become more antibiotic resistant in space microgravity (MG). We report that under low-shear modeled microgravity (LSMMG), stationary-phase uropathogenic Escherichia coli (UPEC) become more resistant to gentamicin (Gm), and that this increase is dependent on the presence of σs (a transcription regulator encoded by the rpoS gene). UPEC causes urinary tract infections (UTIs), reported to afflict astronauts; Gm is a standard treatment, so these findings could impact astronaut health. Because LSMMG findings can differ from MG, we report preparations to examine UPEC's Gm sensitivity during spaceflight using the E. coli Anti-Microbial Satellite (EcAMSat) as a free-flying "nanosatellite" in low Earth orbit. Within EcAMSat's payload, a 48-microwell fluidic card contains and supports study of bacterial cultures at constant temperature; optical absorbance changes in cell suspensions are made at three wavelengths for each microwell and a fluid-delivery system provides growth medium and predefined Gm concentrations. Performance characterization is reported here for spaceflight prototypes of this payload system. Using conventional microtiter plates, we show that Alamar Blue (AB) absorbance changes can assess the Gm effect on E. coli viability, permitting telemetric transfer of the spaceflight data to Earth. Laboratory results using payload prototypes are consistent with wellplate and flask findings of differential sensitivity of UPEC and its ∆rpoS strain to Gm. if σs plays the same role in space MG as in LSMMG and Earth gravity, countermeasures discovered in recent Earth studies (aimed at weakening the UPEC antioxidant defense) to control UPEC infections would prove useful also in space flights. Further, EcAMSat results should clarify inconsistencies from previous space experiments on bacterial antibiotic sensitivity and other issues.

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

BACKGROUND: 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. OBJECTIVES: 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 in situ at -80°C. METHODS: 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). RESULTS: 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. CONCLUSION: 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 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 spaceflight experiments that entail on-orbit sample recovery by astronauts.

Removal of chylomicron remnants in transgenic mice overexpressing normal and membrane-anchored hepatic lipase.

The LDL receptor and the LDL receptor-related protein (LRP) mediate the removal of chylomicron remnants. The LRP pathway involves sequestration of particles in the space of Disse. It has been proposed that either alone or in combination with other factors, such as apolipoprotein E and proteoglycans, hepatic lipase (HL) may contribute to the sequestration of chylomicron remnants. To test this hypothesis, we generated two lines of transgenic mice producing rat HL as a native or as a membrane-anchored form. These animals express HL at levels similar to normal rat. Chylomicron remnants were perfused in a single nonrecirculating pass into the livers of the rat HL transgenic, HL-deficient, and wild-type (WT) mice for 20 min, and the rate of chylomicron remnant removal was measured. Chylomicron remnants were removed at a rate of approximately 50% per pass in WT mice. It was slightly increased in both transgenic mice and reduced in HL-deficient mice compared with the WT mice. Confocal microscopy of liver sections showed that a modest amount of HL colocalized with chylomicron remnant clusters in the transgenic mice, suggesting that HL is a component of the LRP-proteoglycan clusters. These data suggest that HL helps to direct cholesterol to the tissues in which it is localized by a nonenzymatic mechanism.

Association of hepatic lipase with proteoglycans stimulates the production of proteoglycans in vivo and in vitro.

HL is synthesized in hepatocytes and functions while bound to heparan sulfate proteoglycans (HSPGs) in sinusoidal endothelial cells. The HL-mediated uptake of lipoprotein requires cell-surface HSPG. The present study tested whether HL plays a role in the production of HSPG. The production of HSPG in Chinese hamster ovary (CHO) cells was determined by measuring the incorporation of (35)SO(4) into PGs. HL-producing HL-CHO cells showed approximately 30% more cellular PG than did wild-type (WT) cells. In contrast, PG production in cells producing a membrane-anchored HL-glycophosphatidylinositol (GPI) that was not bound to HSPG was virtually identical to that in WT cells. When purified HL was added to the WT- or HL-GPI cells, PG production increased significantly to a level similar to that of the HL-secreting cells, suggesting that the binding of HL to HSPG triggered the increased HSPG production. Heparin reduced PG production in HL-producing cells, confirming that PG production is stimulated only when HL is present as a ligand for HSPG. Real-time PCR and Northern blots demonstrated that PG production was significantly reduced in animals lacking HL. Together, these data suggest that the binding of HL to PG on the cell surface exerts a positive feedback on cellular PG production.

Chylomicron remnant uptake in the livers of mice expressing human apolipoproteins E3, E2 (Arg158-->Cys), and E3-Leiden.

Apolipoprotein E2 (apoE2) and apoE3-Leiden cause chylomicron remnant accumulation (type III hyperlipidemia). However, the degree of dyslipidemia and its penetrance are different in humans and mice. Remnant uptake by isolated liver from apoE-/- mice transgenic for human apoE2, apoE3-Leiden, or apoE3 was measured. In the presence of both LDL receptor (LDLR) and LDL receptor-related protein (LRP), remnant uptake was apoE3>E3-Leiden>E2 mice. Absence of LDLR reduced uptake in apoE3 and apoE3-Leiden-secreting livers but not in apoE2-secreting livers. LRP inhibition with receptor-associated protein reduced uptake in apoE3- and apoE2-secreting livers, but not in apoE3-Leiden-secreting livers, regardless of the presence of LDLR. Fluorescently labeled remnants clustered with LRP in apoE3-secreting livers only in the absence of LDLR, but clustered in livers that expressed apoE2 even in the presence of LDLR, and did not cluster with LRP in livers of apoE3-Leiden even in the absence of LDLR. Remnants were reconstituted with the three human apoE isoforms. Removal by liver of mApoe-/-/mldlr-/- mice expressing the human LDLR was slightly greater than removal in the previous experiments with apoE3>E2> E3-Leiden. Thus, in vivo, human apoE2 is cleared primarily by LRP, apoE3-Leiden is cleared only by the LDLR, and apoE3 is cleared by both.

Endothelial lipase: a new lipase on the block.

Endothelial lipase (EL) is a newly described member of the triglyceride lipase gene family. It has a considerable molecular homology with lipoprotein lipase (LPL) (44%) and hepatic lipase (HL) (41%). Unlike LPL and HL, this enzyme is synthesized by endothelial cells and functions at the site where it is synthesized. Furthermore, its tissue distribution is different from that of LPL and HL. As a lipase, EL has primarily phospholipase A1 activity. Animals that overexpress EL showed reduced HDL cholesterol levels. Conversely, animals that are deficient in EL showed a marked elevation in HDL cholesterol levels, suggesting that it plays a physiologic role in HDL metabolism. Unlike LPL and HL, EL is located in the vascular endothelial cells and its expression is highly regulated by cytokines and physical forces, suggesting that it may play a role in the development of atherosclerosis. However, there is only a limited amount of information available about this enzyme. Some of our unpublished data in addition to previously published data support the possibility that the enzyme plays a role in the formation of atherosclerotic lesion.

Endothelial lipase modulates susceptibility to atherosclerosis in apolipoprotein-E-deficient mice.

Endothelial lipase (EL) expression correlates inversely with circulating high density lipoprotein (HDL) cholesterol levels in genetic mouse models, and human genetic variation in this locus has been linked to differences in HDL cholesterol levels. These data suggest a role for EL in the development of atherosclerotic vascular disease. To investigate this possibility, LIPG-null alleles were bred onto the apoE knockout background, and the homozygous double knockout animals were characterized. Both apoE knockout and double knockout mice had low HDL cholesterol levels when compared with wild-type mice, but the HDL cholesterol levels of the double knockout mice were higher than those of apoE knockout mice. Atherogenic very low density lipoprotein and intermediate density lipoprotein/low density lipoprotein cholesterol levels of the double knockout mice were also greater than those of the apoE knockout animals. Despite this lipid profile, there was a significant approximately 70% decrease in atherosclerotic disease area in double knockout mice on a regular diet. Immunohistochemistry and protein blot studies revealed increased EL expression in the atherosclerotic aortas of the apoE knockout animals. An observed decrease in macrophage content in vessels lacking EL correlated with ex vivo vascular monocyte adhesion assays, suggesting that this protein can modulate monocyte adhesion and infiltration into diseased tissues. These data suggest that EL may have indirect atherogenic actions in vivo through its effect on circulating HDL cholesterol and direct atherogenic actions through vascular wall processes such as monocyte recruitment and cholesterol uptake.

Endothelial lipase is synthesized by hepatic and aorta endothelial cells and its expression is altered in apoE-deficient mice.

Both LPL and HL are synthesized in parenchymal cells, are secreted, and bind to endothelial cells. To learn where endothelial lipase (EL) is synthesized in adult animals, the localization of EL in mouse and rat liver was studied by immunohistochemical analysis. Furthermore, to test whether EL could play a role in atherogenesis, the expression of EL in the aorta and liver of apolipoprotein E knockout (EKO) mice was determined. EL in both mouse and rat liver was colocalized with vascular endothelial cells but not with hepatocytes. In contrast, HL was present in both hepatocytes and endothelial cells. By in situ hybridization, EL mRNA was present only in endothelial cells in liver sections. EL was also present at low levels in aorta of normal mice. We fed EKO mice and wild-type mice a variety of diets and determined EL expression in liver and aorta. EKO mice showed significant expression of EL in aorta. EL expression was lower in the liver of EKO mice than in normal mice. Cholesterol feeding decreased EL in liver of both types of mice. In the aorta, EL was higher in EKO than in wild-type mice, and cholesterol feeding had no effect. Together, these data suggest that EL may be upregulated at the site of atherosclerotic lesions and thus could supply lipids to the area.

Very low density lipoprotein (VLDL) receptor-deficient mice have reduced lipoprotein lipase activity. Possible causes of hypertriglyceridemia and reduced body mass with VLDL receptor deficiency.

Although very low density lipoprotein (VLDL) receptor (VLDLr) knockout mice have been reported to have no lipoprotein abnormalities, they develop less adipose tissue than control mice when fed a high calorie diet. Mice that are deficient in adipose tissue expression of lipoprotein lipase (LpL) also have less fat, but only when crossed with ob/ob mice. We hypothesized that the VLDLr, a protein that will bind and transport LpL, is required for optimal LpL actions in vivo and that hypertriglyceridemia due to VLDLr deficiency is exacerbated by either LpL deficiency or VLDL overproduction. Fasted VLDLr knockout (VLDLr0) mice were more hypertriglyceridemic than controls (2-fold greater triglyceride levels). The hypertriglyceridemia due to VLDLr0 was even more evident when VLDLr0 mice were crossed with heterozygous LpL-deficient (LpL1) and human apolipoprotein B (apoB) transgenic mice. This was due to an increase in apoB48-containing VLDL. [(3)H]VLDL turnover studies showed that VLDL-triglyceride clearance in VLDLr0/LpL1 mice was impaired by 50% compared with LpL1 mice. VLDLr0/LpL1 mice had less LpL activity in postheparin plasma, heart, and skeletal muscle. Infection of mice with an adenovirus-expressing receptor-associated protein, an inhibitor of the VLDLr, reduced LpL activity in wild type but not VLDLr0 mice. Therefore, the VLDLr is required for normal LpL regulation in vivo, and the disruption of VLDLr results in hypertriglyceridemia associated with decreased LpL activity.