To accomplish mission objectives, adult wild-type female mice will be flown to the International Space Station (ISS) using RR Hardware. Groups of mice provided with the NB will be compared to those without the NB in the Rodent Habitat in both Flight and Ground. Comparison of Flight groups (FLT) to their respective Habitat Ground Control (HGC) groups will reveal if and how the NB affects major physiological systems relevant to the discipline of Space Biology.

Preparations for the RR-26 mission have continued during this second grant year, and major mission milestones were achieved. An RR-26 Technical Interchange Meeting (TIM) was held at NASA Ames Research Center in June 2023 to address specific critical topics related to RR-26 science development, operations, and implementation.

A major augmentation of the RR-26 mission involves upgrading existing analog cameras to digital technology, and continuous rather than discrete video sampling of rodent behavior for analysis. In the second year, focus has been on coordinating science requirements with video engineering capabilities.

To support inflight data collection, two studies were conducted to establish the feasibility of utilizing the new Habitat digital cameras to identify individual mice via fur dye markings (Aim 1), continuous body temperature acquired using implanted dataloggers (Aim 2), and assessment of the circadian timing system using video and body temperature data to assess parallel changes across light and dark cycle phases (Aim 3). Due to the current technical inability to measure mouse sleep on the International Space Station/ISS (Aim 4), we sought proof-of-concept that sleep-like behavior can be inferred from these circadian-sensitive measures. Aims 5-9, critical for addressing the role(s) of the Nest Box (NB) in brain, bone, muscle, immune, and cardiovascular responses to spaceflight, require little or no preliminary testing owing to numerous past spaceflight studies. For these reasons, only Science Tests supporting RR-26 Aims 1-4 are described in the Test Plan Document No. 8020, RR26 Science Tests to Validate Fur Bleach Patterns and Datalogger Implants in Mice.

RESULTS

ST-1 and -3 Main Findings Supported Aims 1-4. Objectives were successfully met as described here:

ST-1 • The Launch Simulation Test successfully validated that datalogger implantation did not damage or elicit concerning pathological responses in the mouse abdominal cavity (NASA Attending Vet Necropsy report). Continuous acquisition of datalogger temperature measures was reliable, and unaffected by launch forces. • Datalogger use in the Habitat and under launch simulation conditions has been successfully vetted and established with zero concerns.

ST-3 • Individual identification from fur dye patterns in combination with ear notches is attainable within the RR-26 habitat with a full complement of mice (5 per side). In this study, fur dyes were readable for more than two weeks. • Optimization tests conducted by Dr. Alberts at Indiana University have since extended fur durability duration to the 30-day mission length. • The video system in its current configuration is adequate. It will provide “first ever” views and data, and thus can make substantial contributions to RR-26 science and to Space Biology more generally. However, there is much room for improvement, and we strongly support NASA in further developing this system using available and affordable technologies. • Preliminary analysis of huddling behavior relative to body temperature suggests the possibility of identifying meaningful relations between these measures; however, analysis was limited by short duration video samples.

CONCLUSION

The data collected from the implanted dataloggers will provide continuous measurement of the body temperature of each animal throughout the RR-26 mission, beginning with preflight control measurements. These data are integral for the peer-reviewed outcomes of Specific Aims 2 & 3. For these Aims, we will utilize the body temperature data provided by the data loggers to determine how the Circadian Timing System responds and adapts to the spaceflight environment. Moreover, the data will be used to understand metabolism and temperature regulation during the mission. These data will also be extremely valuable to other investigators. Data from the loggers will provide a direct physiological indication of the overall health and well-being of the subjects, as well as each animal’s adaptation pattern to microgravity, over the course of the mission. Given robust segments of continuous video acquired simultaneously from each habitat’s camera set, changes in body temperature data can also be analyzed in combination with the recorded behavioral observations to bolster information on the rest-activity patterns of each animal as surrogate measures of their sleep wake regulation. This is especially important as we cannot, currently, record EEG to measure sleep/wake directly. Finally, we will be able to, for the first time, make direct correlations between a physiological variable and an animal’s individual and group behaviors.

To accomplish mission objectives, adult wild-type female mice will be flown to the International Space Station (ISS) using RR Hardware. Groups of mice provided with the NB will be compared to those without the NB in the Rodent Habitat in both Flight and Ground. Comparison of Flight groups (FLT) to their respective Habitat Ground Control (HGC) groups will reveal if and how the NB affects major physiological systems relevant to the discipline of Space Biology.

To accomplish mission objectives, adult wild-type female mice will be flown to the International Space Station (ISS) using RR Hardware. Groups of mice provided with the NB will be compared to those without the NB in the Rodent Habitat in both Flight and Ground. Comparison of Flight groups (FLT) to their respective Habitat Ground Control (HGC) groups will reveal if and how the NB affects major physiological systems relevant to the discipline of Space Biology.