Evidence of Spaceflight-Induced Adverse Effects on Photoreceptors and Retinal Function in the Mouse Eye.

The goal of the present study was to characterize acute oxidative damage in ocular structure and retinal function after exposure to spaceflight, and to evaluate the efficacy of an antioxidant in reducing spaceflight-induced changes in the retina. Ten-week-old adult C57BL/6 male mice were flown aboard the ISS on Space-X 24 over 35 days, and returned to Earth alive. The mice received a weekly injection of a superoxide dismutase mimic, MnTnBuOE-2-PyP 5+ (BuOE), before launch and during their stay onboard the ISS. Ground control mice were maintained on Earth under identical environmental conditions. Before the launch, intraocular pressure (IOP) was measured using a handheld tonometer and retinal function was evaluated using electroretinogram (ERG). ERG signals were recorded when the mouse eye was under dark-adapted conditions in response to ultraviolet monochromatic light flashes. Within 20 h after splashdown, IOP and ERG assessments were repeated before euthanasia. There were significant increases in body weight for habitat control groups post-flight compared to pre-flight measurements. However, the body weights were similar among flight groups before launch and after splashdown. The IOP measurements were similar between pre- and post-flight groups with no significant differences between BuOE-treated and saline controls. Immunofluorescence evaluation showed increases in retinal oxidative stress and apoptotic cell death after spaceflight. BuOE treatment significantly decreased the level of the oxidative stress biomarker. ERG data showed that the average amplitudes of the a- and b-wave were significantly decreased (39% and 32% by spaceflight, respectively) compared to that of habitat ground controls. These data indicate that spaceflight conditions induce oxidative stress in the retina, which may lead to photoreceptor cell damage and retinal function impairment.

Spaceflight-Induced Gene Expression Profiles in the Mouse Brain Are Attenuated by Treatment with the Antioxidant BuOE.

The demands of deep space pose a health risk to the central nervous system that has long been a concern when sending humans to space. While little is known about how spaceflight affects transcription spatially in the brain, a greater understanding of this process has the potential to aid strategies that mitigate the effects of spaceflight on the brain. Therefore, we performed GeoMx Digital Spatial Profiling of mouse brains subjected to either spaceflight or grounded controls. Four brain regions were selected: Cortex, Frontal Cortex, Corunu Ammonis I, and Dentate Gyrus. Antioxidants have emerged as a potential means of attenuating the effects of spaceflight, so we treated a subset of the mice with a superoxide dismutase mimic, MnTnBuOE-2-PyP 5+ (BuOE). Our analysis revealed hundreds of differentially expressed genes due to spaceflight in each of the four brain regions. Both common and region-specific transcriptomic responses were observed. Metabolic pathways and pathways sensitive to oxidative stress were enriched in the four brain regions due to spaceflight. These findings enhance our understanding of brain regional variation in susceptibility to spaceflight conditions. BuOE reduced the transcriptomic effects of spaceflight at a large number of genes, suggesting that this compound may attenuate oxidative stress-induced brain damage caused by the spaceflight environment.

Spaceflight induces oxidative damage to blood-brain barrier integrity in a mouse model.

Many factors contribute to the health risks encountered by astronauts on missions outside Earth's atmosphere. Spaceflight-induced potential adverse neurovascular damage and late neurodegeneration are a chief concern. The goal of the present study was to characterize the effects of spaceflight on oxidative damage in the mouse brain and its impact on blood-brain barrier (BBB) integrity. Ten-week-old male C57BL/6 mice were launched to the International Space Station (ISS) for 35 days as part of Space-X 12 mission. Ground control (GC) mice were maintained on Earth in flight hardware cages. Within 38 ± 4 hours after returning from the ISS, mice were euthanized and brain tissues were collected for analysis. Quantitative assessment of brain tissue demonstrated that spaceflight caused an up to 2.2-fold increase in apoptosis in the hippocampus compared to the control group. Immunohistochemical analysis of the mouse brain revealed an increased expression of aquaporin4 (AQP4) in the flight hippocampus compared to the controls. There was also a significant increase in the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and a decrease in the expression of the BBB-related tight junction protein, Zonula occludens-1 (ZO-1). These results indicate a disturbance of BBB integrity. Quantitative proteomic analysis showed significant alterations in pathways responsible for neurovascular integrity, mitochondrial function, neuronal structure, protein/organelle transport, and metabolism in the brain after spaceflight. Changes in pathways associated with adhesion and molecular remodeling were also documented. These data indicate that long-term spaceflight may have pathological and functional consequences associated with neurovascular damage and late neurodegeneration.

Mesenchymal-endothelial transition contributes to cardiac neovascularization.

Endothelial cells contribute to a subset of cardiac fibroblasts by undergoing endothelial-to-mesenchymal transition, but whether cardiac fibroblasts can adopt an endothelial cell fate and directly contribute to neovascularization after cardiac injury is not known. Here, using genetic fate map techniques, we demonstrate that cardiac fibroblasts rapidly adopt an endothelial-cell-like phenotype after acute ischaemic cardiac injury. Fibroblast-derived endothelial cells exhibit anatomical and functional characteristics of native endothelial cells. We show that the transcription factor p53 regulates such a switch in cardiac fibroblast fate. Loss of p53 in cardiac fibroblasts severely decreases the formation of fibroblast-derived endothelial cells, reduces post-infarct vascular density and worsens cardiac function. Conversely, stimulation of the p53 pathway in cardiac fibroblasts augments mesenchymal-to-endothelial transition, enhances vascularity and improves cardiac function. These observations demonstrate that mesenchymal-to-endothelial transition contributes to neovascularization of the injured heart and represents a potential therapeutic target for enhancing cardiac repair.

Mice Exposed to Combined Chronic Low-Dose Irradiation and Modeled Microgravity Develop Long-Term Neurological Sequelae.

Spaceflight poses many challenges for humans. Ground-based analogs typically focus on single parameters of spaceflight and their associated acute effects. This study assesses the long-term transcriptional effects following single and combination spaceflight analog conditions using the mouse model: simulated microgravity via hindlimb unloading (HLU) and/or low-dose γ-ray irradiation (LDR) for 21 days, followed by 4 months of readaptation. Changes in gene expression and epigenetic modifications in brain samples during readaptation were analyzed by whole transcriptome shotgun sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS). The results showed minimal gene expression and cytosine methylation alterations at 4 months readaptation within single treatment conditions of HLU or LDR. In contrast, following combined HLU+LDR, gene expression and promoter methylation analyses showed multiple altered pathways involved in neurogenesis and neuroplasticity, the regulation of neuropeptides, and cellular signaling. In brief, neurological readaptation following combined chronic LDR and HLU is a dynamic process that involves pathways that regulate neuronal function and structure and may lead to late onset neurological sequelae.

Occurrences of yawn and swallow are temporally related.

Yawning is a stereotyped motor behavior characterized by deep inhalation and associated dilation of the respiratory tract, pronounced jaw opening, and facial grimacing. The frequency of spontaneous yawning varies over the diurnal cycle, peaking after waking and before sleep. Yawning can also be elicited by seeing or hearing another yawn, or by thinking about yawning, a phenomenon known as "contagious yawning". Yawning is mediated by a distributed network of brainstem and supratentorial brain regions, the components of which are shared with other airway behaviors including respiration, swallowing, and mastication. Nevertheless, the possibility of behavioral coordination between yawning and other brainstem-mediated functions has not been examined. Here we show, with a double-blind methodology, a greater-than-fivefold increase in rest (saliva) swallowing rate during the 10-s period immediately following contagious yawning elicited in 14 adult humans through the viewing of videotaped yawn stimuli. Sixty-five percent of yawns were followed by a swallow within 10 s and swallows accounted for 26 % of all behaviors produced during this post-yawn period. This novel finding of a tight temporal coupling between yawning and swallowing provides preliminary evidence that yawning and swallowing are physiologically related, thus extending current models of upper airway physiology and neurophysiology. Moreover, our finding suggests the possibility that yawning plays a role in eliciting rest swallowing, a view not considered in previous theories of yawning. As such, the present demonstration of a temporal association between yawning and swallowing motivates a re-examination of the longstanding question, "Why do we yawn?".

Characterization of gene expression profiles in the mouse brain after 35 days of spaceflight mission.

It has been proposed that neuroinflammatory response plays an important role in the neurovascular remodeling in the brain after stress. The goal of the present study was to characterize changes in the gene expression profiles associated with neuroinflammation, neuronal function, metabolism and stress in mouse brain tissue. Ten-week old male C57BL/6 mice were launched to the International Space Station (ISS) on SpaceX-12 for a 35-day mission. Within 38 ± 4 h of splashdown, mice were returned to Earth alive. Brain tissues were collected for analysis. A novel digital color-coded barcode counting technology (NanoStringTM) was used to evaluate gene expression profiles in the spaceflight mouse brain. A set of 54 differently expressed genes (p < 0.05) significantly segregates the habitat ground control (GC) group from flight (FLT) group. Many pathways associated with cellular stress, inflammation, apoptosis, and metabolism were significantly altered by flight conditions. A decrease in the expression of genes important for oligodendrocyte differentiation and myelin sheath maintenance was observed. Moreover, mRNA expression of many genes related to anti-viral signaling, reactive oxygen species (ROS) generation, and bacterial immune response were significantly downregulated. Here we report that significantly altered immune reactions may be closely associated with spaceflight-induced stress responses and have an impact on the neuronal function.

Evaluating Ocular Response in the Retina and Optic Nerve Head after Single and Fractionated High-Energy Protons.

There are serious concerns about possible late radiation damage to ocular tissue from prolonged space radiation exposure, and occupational and medical procedures. This study aimed to investigate the effects of whole-body high-energy proton exposure at a single dose on apoptosis, oxidative stress, and blood-retina barrier (BRB) integrity in the retina and optic nerve head (ONH) region and to compare these radiation-induced effects with those produced by fractionated dose. Six-month-old C57BL/6 male mice were either sham irradiated or received whole-body high energy proton irradiation at an acute single dose of 0.5 Gy or 12 equal dose fractions for a total dose of 0.5 Gy over twenty-five days. At four months following irradiation, mice were euthanized and ocular tissues were collected for histochemical analysis. Significant increases in the number of apoptotic cells were documented in the mouse retinas and ONHs that received proton radiation with a single or fractionated dose (p < 0.05). Immunochemical analysis revealed enhanced immunoreactivity for oxidative biomarker, 4-hydroxynonenal (4-HNE) in the retina and ONH following single or fractionated protons with more pronounced changes observed with a single dose of 0.5 Gy. BRB integrity was also evaluated with biomarkers of aquaporin-4 (AQP-4), a water channel protein, a tight junction (TJ) protein, Zonula occludens-1 (ZO-1), and an adhesion molecule, the platelet endothelial cell adhesion molecule-1 (PECAM-1). A significantly increased expression of AQP-4 was observed in the retina following a single dose exposure compared to controls. There was also a significant increase in the expression of PECAM-1 and a decrease in the expression of ZO-1 in the retina. These changes give a strong indication of disturbance to BRB integrity in the retina. Interestingly, there was very limited immunoreactivity of AQP-4 and ZO-1 seen in the ONH region, pointing to possible lack of BRB properties as previously reported. Our data demonstrated that exposure to proton radiation of 0.5 Gy induced oxidative stress-associated apoptosis in the retina and ONH, and changes in BRB integrity in the retina. Our study also revealed the differences in BRB biomarker distribution between these two regions. In response to radiation insults, the cellular response in the retina and ONH may be differentially regulated in acute or hyperfractionated dose schedules.

Spaceflight decelerates the epigenetic clock orchestrated with a global alteration in DNA methylome and transcriptome in the mouse retina.

Astronauts exhibit an assortment of clinical abnormalities in their eyes during long-duration spaceflight. The purpose of this study was to determine whether spaceflight induces epigenomic and transcriptomic reprogramming in the retina or alters the epigenetic clock. The mice were flown for 37 days in animal enclosure modules on the International Space Station; ground-based control animals were maintained under similar housing conditions. Mouse retinas were isolated and both DNA methylome and transcriptome were determined by deep sequencing. We found that a large number of genes were differentially methylated with spaceflight, whereas there were fewer differentially expressed genes at the transcriptome level. Several biological pathways involved in retinal diseases such as macular degeneration were significantly altered. Our results indicated that spaceflight decelerated the retinal epigenetic clock. This study demonstrates that spaceflight impacts the retina at the epigenomic and transcriptomic levels, and such changes could be involved in the etiology of eye-related disorders among astronauts.

Establishing community reference samples, data and call sets for benchmarking cancer mutation detection using whole-genome sequencing.

The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses.

Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography (Micro-CT) Imaging Method.

Reports show that prolonged exposure to a spaceflight environment produces morphologic and functional ophthalmic changes in astronauts during and after an International Space Station (ISS) mission. However, the underlying mechanisms of these spaceflight-induced changes are currently unknown. The purpose of the present study was to determine the impact of the spaceflight environment on ocular structures by evaluating the thickness of the mouse retina, the retinal pigment epithelium (RPE), the choroid and the sclera layer using micro-CT imaging. Ten-week-old C57BL/6 male mice were housed aboard the ISS for a 35-day mission and then returned to Earth alive for tissue analysis. For comparison, ground control (GC) mice on Earth were maintained in identical environmental conditions and hardware. Ocular tissue samples were collected for micro-CT analysis within 38(±4) hours after splashdown. The images of the cross-section of the retina, the RPE, the choroid, and the sclera layer of the fixed eye was recorded in an axial and sagittal view using a micro-CT imaging acquisition method. The micro-CT analysis showed that the cross-section areas of the retina, RPE, and choroid layer thickness were changed in spaceflight samples compared to GC, with spaceflight samples showing significantly thinner cross-sections and layers compared to controls. The findings from this study indicate that micro-CT evaluation is a sensitive and reliable method to characterize ocular structure changes. These results are expected to improve the understanding of the impact of environmental stress on global ocular structures.

Spaceflight influences gene expression, photoreceptor integrity, and oxidative stress-related damage in the murine retina.

Extended spaceflight has been shown to adversely affect astronaut visual acuity. The purpose of this study was to determine whether spaceflight alters gene expression profiles and induces oxidative damage in the retina. Ten week old adult C57BL/6 male mice were flown aboard the ISS for 35 days and returned to Earth alive. Ground control mice were maintained on Earth under identical environmental conditions. Within 38 (+/-4) hours after splashdown, mice ocular tissues were collected for analysis. RNA sequencing detected 600 differentially expressed genes (DEGs) in murine spaceflight retinas, which were enriched for genes related to visual perception, the phototransduction pathway, and numerous retina and photoreceptor phenotype categories. Twelve DEGs were associated with retinitis pigmentosa, characterized by dystrophy of the photoreceptor layer rods and cones. Differentially expressed transcription factors indicated changes in chromatin structure, offering clues to the observed phenotypic changes. Immunofluorescence assays showed degradation of cone photoreceptors and increased retinal oxidative stress. Total retinal, retinal pigment epithelium, and choroid layer thickness were significantly lower after spaceflight. These results indicate that retinal performance may decrease over extended periods of spaceflight and cause visual impairment.

Characterization of mouse ocular response to a 35-day spaceflight mission: Evidence of blood-retinal barrier disruption and ocular adaptations.

The health risks associated with spaceflight-induced ocular structural and functional damage has become a recent concern for NASA. The goal of the present study was to characterize the effects of spaceflight and reentry to 1 g on the structure and integrity of the retina and blood-retinal barrier (BRB) in the eye. To investigate possible mechanisms, changes in protein expression profiles were examined in mouse ocular tissue after spaceflight. Ten week old male C57BL/6 mice were launched to the International Space Station (ISS) on Space-X 12 at the Kennedy Space Center (KSC) on August, 2017. After a 35-day mission, mice were returned to Earth alive. Within 38 +/- 4 hours of splashdown, mice were euthanized and ocular tissues were collected for analysis. Ground control (GC) and vivarium control mice were maintained on Earth in flight hardware or normal vivarium cages respectively. Repeated intraocular pressure (IOP) measurements were performed before the flight launch and re-measured before the mice were euthanized after splashdown. IOP was significantly lower in post-flight measurements compared to that of pre-flight (14.4-19.3 mmHg vs 16.3-20.3 mmHg) (p < 0.05) for the left eye. Flight group had significant apoptosis in the retina and retinal vascular endothelial cells compared to control groups (p < 0.05). Immunohistochemical analysis of the retina revealed that an increased expression of aquaporin-4 (AQP-4) in the flight mice compared to controls gave strong indication of disturbance of BRB integrity. There were also a significant increase in the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and a decrease in the expression of the BRB-related tight junction protein, Zonula occludens-1 (ZO-1). Proteomic analysis showed that many key proteins and pathways responsible for cell death, cell cycle, immune response, mitochondrial function and metabolic stress were significantly altered in the flight mice compared to ground control animals. These data indicate a complex cellular response that may alter retina structure and BRB integrity following long-term spaceflight.

Sodium hydrogen exchange 1 (NHE-1) regulates connexin 43 expression in cardiomyocytes via reverse mode sodium calcium exchange and c-Jun NH2-terminal kinase-dependent pathways.

Connexin 43, the major connexin isoform in gap junctions of cardiac ventricular myocytes, undergoes changes in distribution and expression in cardiac diseases. The Na(+)-H(+) exchanger (NHE-1), a key mediator of hypertrophy and heart failure, has been shown to be localized in the cardiomyocyte gap junctional regions; however, whether NHE-1 regulates gap junction proteins in the hypertrophied cardiomyocyte is not known. To address this question, neonatal rat ventricular myocytes were treated with phenylephrine (PE) for 24 h to induce hypertrophy. Increased Cx43 expression observed with PE treatment (132.4 +/- 6.3% compared to control; P < 0.05) was further significantly augmented by the specific NHE-1 inhibitor EMD87580 [N-[2-methyl-4,5-bis(methylsulfonyl)-benzoyl]-guanidine hydrochloride] (173.2 +/- 8.7% increase compared to control; P < 0.05 versus PE), an effect that was mimicked by another NHE-1 inhibitor cariporide [4-isopropyl-3-(methylsulfonyl)benzoyl-guanidine methanesulfonate]. PE-induced hypertrophy was associated with mitogen-activated protein kinase c-Jun NH(2)-terminal kinase (JNK) 1/2 activation, whereas inhibition of JNK1/2 with either SP600125 [anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone] or small interfering RNA significantly increased PE-induced up-regulation of Cx43 protein levels. Inhibition of reverse mode Na(+)-Ca(2+) exchange (NCX) with KB-R7943 [2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate] partially reversed JNK1/2 activation (195.2 +/- 21.4 versus 143.7 +/- 14.4% with KB-R7943; P < 0.05) and augmented up-regulation of Cx43 protein (121.1 +/- 8.3 versus 215.9 +/- 25.6% with KB-R7943; P < 0.05) in the presence of PE. Our results demonstrate that NHE-1 negatively regulates Cx43 protein expression in PE-induced cardiomyocyte hypertrophy via a JNK1/2-dependent pathway, which is probably activated by reverse mode NCX activity.