In recent years, there has been significant advancement in microgravity experiments facilitated by using the International Space Station (ISS). Multiple microgravity experiments have been conducted aboard ISS Kibo, while the Japan Aerospace Exploration Agency (JAXA) has successfully designed a novel mouse cage specifically tailored for space studies. The practice of animal breeding is necessary to examine gravity's impact on animal development and get insights into the mechanisms behind adaptation to partial gravity settings. The study of animal reproduction on extraterrestrial bodies and in space is crucial for comprehending the mechanisms behind environmental adaptability. 
 The aging process of mice in space is seen to be around 10 to 30 times more rapid on the timeline compared to their aging process on Earth. In the old age of human life, the muscles of the bladder and the whole body undergo aging, resulting in impaired urinary function and necessitating the use of an indwelling catheter for some individuals. The condition is missing targeted therapy, which is a leading cause of lower quality of life. Therefore, a prompt investigation into its etiology and the search for a cure for lower urinary tract symptoms (LUTS), especially detrusor underactivity, is desired. The first mouse project in space was carried out by JAXA in 2016. A centrifuge-equipped biological experiment equipment was used to generate microgravity (MG) and artificial 1G (AG) in space [1]. The mouse bladder was successfully sample-shared, enabling further analysis of RNA-seq data. The objective of this analysis is to initially determine the molecular mechanism underlying the issue involved in urination impairment in space at the level of gene expression and subsequently utilize this mechanistic information to develop a treatment for the problem regarding LUTS in a space environment and aging.

The study included the rearing of twelve male C57BL/6 mice in a controlled environment with either MG or AG. The mice were divided into two groups (six mice in MG and six mice in AG) [1]. MG mice were suspended in the biological laboratory facility, and AG mice had their feet on the floor. After 35 days of habitation on the ISS, all mice were returned to Earth and processed. The dragon capsule containing the rodents was subsequently touched down in the Pacific Ocean near the West Coast. Two days later, mice were transported to the laboratory in order to undergo behavioral observation and dissection. Six ground-gravity mice (NG) were used as controls. The bladder tissues of mice were formalin-fixed paraffin-embedded (FFPE). RNA-seq was conducted using the extracted RNA from these FFPE tissues.

KEGG pathway and GO analyses were performed using genes that met filter criteria (> 1.75 or < - 1.75-fold change, P < 0.05). A total of 434 transcripts exhibited variable expression between MG and NG, 231 between AG and MG, and 395 between AG and NG. KEGG Pathway and GO analysis, using differentially expressed genes between MG and NG conditions, also identified genes related to circadian rhythm (Fig. 1 and 2). The GO analysis conducted in the comparison between MG and NG revealed alterations in circadian rhythm-related, muscle differentiation-related hemoglobin, aerobic respiration, and erythrocyte-related factors under the Biological Process (BP) category. Additionally, alterations were observed in hemoglobin, aerobic respiration, and erythrocyte-related factors within the Cellular Component (CC) and Molecular Function (MF) categories (Fig.2). The GO analysis conducted on AG vs. NG revealed alterations in hemoglobin levels, aerobic respiration, and erythrocyte association within the CC, BP, and MF domains. The GO analysis in AG vs. MG revealed alterations in sodium ion transport and genes linked to muscle contraction in BP and in genes related to muscle contraction in CC.

KEGG Pathway and GO analysis identified genes related to circadian rhythm. Most cells and organs contain clock genes, which regulate sleep-wake cycles. According to previous reports, the bladder mucosa of mice exhibits a circadian rhythm. Specifically, the Per2 gene is significantly expressed in the bladder mucosa during the light cycle, which relates to the sleep phase. In contrast, the Bmal1 gene demonstrates substantial expression during the opposite dark cycle, which corresponds to the active phase [2]. Despite obtaining the current sample from a whole bladder, we anticipate that the upregulation of Bmal1 expression and the downregulation of Per2 expression during weightlessness could potentially facilitate pollakiuria.
The GO analysis in AG vs. MG revealed alterations in sodium ion transport. Reports have shown that mice in space transfer sodium and water from their lower limb skin to their rear side skin [3.] Given this, it's plausible that the transportation of sodium and water to the posterior region may have impacted the bladder after applying artificial gravity, thereby triggering responses from the gene cluster responsible for sodium ion transport. It is possible that the edema experienced by astronauts is not solely attributable to water movement in the lower extremities' epidermis but also includes water from the urine.
 The results of MG vs. NG and AG vs. MG indicate a variable group of muscle contraction-related genes. Skeletal muscle degradation due to weightlessness is well-documented. Some astronauts are reportedly unable to urinate effectively in zero-gravity environments; therefore, they receive CIC (Clean Intermittent Catheterization) training before takeoff. Although it is possible that microgravity might accelerate the atrophy and degradation of smooth muscle in the bladder, additional research on histopathology is required to confirm this.

This is the first report of an investigation into the bladder function of mice in microgravity in space. Additional fundamental research, with a particular focus on excretion management, is necessary to establish a sustainable society in space and improve the quality of life affected by age-related urination impairment.

Development of new experimental platform 'MARS'-Multiple Artificial-gravity Research System-to elucidate the impacts of micro/partial gravity on mice. Sci Rep. 2017;7:10837.
Clock Genes Regulate the Circadian Expression of Piezo1, TRPV4, Connexin26, and VNUT in an Ex Vivo Mouse Bladder Mucosa. PLoS One. 2017;12:e0168234.
Blood pressure adaptation in vertebrates: comparative biology. Kidney Int. 2022;102:242-247.

Continence 12S (2024) 101623
DOI: 10.1016/j.cont.2024.101623