Space exploration exposes humans to microgravity, radiation, altered gas composition, stress, and isolation. To deeply characterize the molecular response to these factors, longitudinal transcriptomic profiles are needed. This study utilizes exosomes, nanoscale vesicles released by all cells and found in body fluids (urine, plasma, CSF), which contain molecular genetic material, including the RNA transcriptome. This allows monitoring of tissue-based changes from easily accessible fluids. The focus is on Spaceflight-Associated Neuro-ocular Syndrome (SANS), characterized by ocular structural and visual manifestations, including optic disc edema, optic nerve sheath distension, increased retinal and choroidal thickness, and posterior globe flattening. While elevated intracranial pressure (ICP) is hypothesized to be associated with SANS, the exact relationship remains unclear. Genetic factors also play a role. This study aims to characterize SANS risk through longitudinal transcriptomic profiling of astronauts, using the exosome-based approach. Idiopathic intracranial hypertension (IIH), a terrestrial analog with similar characteristics to SANS, is used in a pilot phase to test the technology.

Previous research has shown that a terrestrial analog of increased ICP is characterized by inflammatory transcriptional signatures in the CSF of affected individuals through the analysis of exosomes. Most exosome RNA studies focus on small RNAs (miRNAs), but this study investigates a broader diversity of long RNAs, achieving similar diversity as tissue sequencing. Other studies have explored the use of extracellular vesicles in various contexts, like assessing the impact of microgravity on endothelial cells and their RNA transcriptome, and employing urine exosome RNA analysis for prostate cancer diagnosis. However, studies applying exosome technology to space biology are scarce, with some retrospective investigations showing changes in mt-DNA levels in astronauts' plasma after short space missions.

The study involved collecting whole blood and plasma before and after lumbar puncture (LP) in IIH patients with elevated ICP. CSF was collected at the first visit, and urine at a follow-up visit. EVs were isolated from plasma, urine, and CSF, followed by RNA extraction. Intracellular RNA was also isolated from whole blood. Total RNA-Seq libraries were created, and gene expression analysis performed. The quality of sequencing from EV RNA in different biofluids was assessed, comparing the proportion of mappable reads and the number of genes detected. Differential expression analysis was conducted to identify differentially expressed genes between pre- and post-treatment samples in whole blood and plasma. Gene set enrichment analysis (GSEA) was performed to identify pathways affected by high ICP. A comparison was made between CSF samples from patients with high ICP and control samples from a biobank. The compatibility of the NASA in-flight urine collection tubes with the whole transcriptome profiling technology was tested. Finally, the feasibility of preserving cDNA libraries in dry form for long-term storage was evaluated using three commercially available preservation agents (DNAstable™, Whatman™ FTA elute card, and GenTegra-DNA).

The study found that the quality of RNA-Seq data varied across biofluids, with whole blood showing more intron reads due to unprocessed transcripts. Plasma EVs provided more diverse gene expression information compared to whole blood, enabling better biomarker discovery. In IIH patients, 185 differentially expressed genes were found in plasma EVs after treatment, compared to only three in whole blood. GSEA showed that upregulated genes post-treatment were related to adhesion and extracellular structures, while downregulated genes were involved in immune response. In CSF from patients with high ICP, 777 genes were upregulated and 134 downregulated compared to normal controls, with a strong impact on immune-related genes. Urine samples from post-treatment patients showed fewer significant gene expression changes compared to normal controls. The NASA Sarstedt urine collection tubes proved compatible with the exosome RNA isolation method. Finally, cDNA libraries could be stored desiccated at room temperature for at least one month without significant loss of quality, with DNAstable™ being the most effective preservation agent.

This study demonstrates the feasibility of using an exosome-based platform for longitudinal molecular monitoring of astronauts' health during long-duration space missions. The choice of biofluid depends on the research question, with plasma offering systemic information and urine providing data enriched for the urogenital system. CSF, while having less RNA, is enriched for brain and immune system genes. The finding that the NASA urine collection tubes are compatible with the technology opens up possibilities for retrospective studies using already collected samples. The ability to store cDNA libraries in dry form at room temperature significantly simplifies storage and transportation in space, facilitating long-term health monitoring. The use of exosomes in this context is novel for space biology research and shows potential applications beyond space exploration for other medical and occupational settings.

This proof-of-concept study establishes a novel exosome-based approach for monitoring the molecular changes associated with spaceflight-induced conditions, specifically SANS and high ICP. The findings highlight the utility of plasma and urine as readily accessible sources of information, the compatibility of existing in-flight urine collection systems with the technology, and the feasibility of preserving RNA data for long-term storage. This approach holds promise for longitudinal health surveillance of astronauts and has potential implications for other areas of medicine and occupational health.

The study's sample size was small, particularly for the IIH patient group, potentially limiting the generalizability of the findings. The analysis focused on IIH patients as a model for SANS, which may not perfectly capture all aspects of the spaceflight condition. Further research with larger sample sizes, including astronauts during long duration spaceflights, is needed to confirm these findings and better understand the role of exosomal RNA changes in SANS.