NOTE: End date changed to 5/31/2021 per F. Hernandez/ARC and NSSC information (Ed., 5/5/21)

NOTE: End date changed to 3/31/2021 per F. Hernandez/ARC (Ed., 4/7/2020)

NOTE: End date changed to 3/31/2020 per F. Hernandez/ARC (Ed., 6/23/17)

To study these problems, we will determine the hip and knee joint damage that occurs in mice that will fly in space on the International Space Station for 30 days. This damage will be compared to the hip and knee joint damage in another group of mice kept on Earth that also will not have weight on the hip and knee joints for 30 days, with or without receiving radiation exposure that simulates a solar flare. Damage to the hip and knee joint structures will be determined using imaging techniques, engineering devices to measure tissue strength, stained tissue sections, and identification of the molecules that cause the damage. The ability to walk normally after 30 days of weightlessness will also be determined. Finally, we will determine if treadmill running or climbing can reverse any of the hip and knee joint damage caused by being in the weightless space environment.

Our goal is to determine, 1] if hip and knee joint damage occurs in the weightless space environment, and 2] if recovery from this damage is possible with exercise.

With a return to lab from COVID restrictions, we compiled and wrote a manuscript that incorporated all the finished data from our ISS mission, together with the parallel ground-based tail suspension study (with and without a readaptation period that included exercise interventions or not), and also included data from knee joint degradation that were collected during the final Space Shuttle mission (STS-135). Our final conclusion was that spaceflight and reduced weight-bearing on the ground induce an arthritic response in the knees of mice, which can be reversed with a return to weight-bearing (after hindlimb unloading-HLU) if exercise is performed. This final paper was submitted to Scientific Reports and published in the fall. Moreover, the student who was working on the project as his dissertation research, Andy Kwok, wrote and defended his dissertation in Spring, 2021. [Ed. note: see Bibliography section for noted publications]

ANNUAL REPORTING SEPTEMBER 2020

We examined if reduced weight bearing on Earth using a hindlimb unloading (HU) model could cause damage to menisci and cartilage, as during spaceflight. What we found was that in male and female mice, the degree of cartilage and meniscus damage was similar to what was observed after 30 days in space, and occurred at the same location (the region in the knee that supports most of the weight versus gravity). Thus cartilage degradation, and meniscal loss, occurs with reduced weight bearing, and HU is a good analogue for joint damage that occurs during spaceflight.

We then identified that both running and climbing exercise can in part mitigate the damage to cartilage and menisci that is caused by reduced weight bearing.

NOTE: End date changed to 3/31/2021 per F. Hernandez/ARC (Ed., 4/7/2020)

NOTE: End date changed to 3/31/2020 per F. Hernandez/ARC (Ed., 6/23/17)

To study these problems, we will determine the hip and knee joint damage that occurs in mice that will fly in space on the International Space Station for 30 days. This damage will be compared to the hip and knee joint damage in another group of mice kept on Earth that also will not have weight on the hip and knee joints for 30 days, with or without receiving radiation exposure that simulates a solar flare. Damage to the hip and knee joint structures will be determined using imaging techniques, engineering devices to measure tissue strength, stained tissue sections, and identification of the molecules that cause the damage. The ability to walk normally after 30 days of weightlessness will also be determined. Finally, we will determine if treadmill running or climbing can reverse any of the hip and knee joint damage caused by being in the weightless space environment.

Our goal is to determine, 1] if hip and knee joint damage occurs in the weightless space environment, and 2] if recovery from this damage is possible with exercise.

We have met 3 main milestones during the past year. i] We have completed the ground-based Definition Phase portion for Specific Aim 1 and have published that portion in the journal Radiation Research, which examines the combined effect of reduced weight-bearing (HLU--hindlimb unloading) with/without exposure to one of 3 low dose radiation scenarios (0.1, 0.5, or 1 Gy) in regards to whole joint health. ii] We have completed the flight experiment. The data from the gait assessment has been submitted for publication; we have resubmitted our revisions. iii] Additionally, we have finished analyzing the spaceflight data and have started writing up a publication (combining these results with results from STS-135).

Progress to date includes:

A. Results from Specific Aim 1 Definition Phase – a ground based study in which we examined the individual and combined effects of low dose radiation and reduced weight bearing via tail suspension in mice: Both reduced weight bearing and low dose radiation as independent challenges cause degradation of knee cartilage and increases arthritic, catabolic signaling pathways in cartilage that can lead to arthritis.

B. Flight study RR-9: Our mouse payload has been launched as part of the Rodent Research-9 mission aboard SpaceX-12 to the International Space Station. Our two primary aims were to examine if knee joint degradation (e.g., articular cartilage and menisci) occurred after spaceflight; and if gait alterations occurred that could indicate pathology in the joint or otherwise.

B1. Knee joint findings. Articular cartilage volume lining the medial tibial plateau in FLIGHT mice measured from microCT was significantly lower than controls. Cartilage thinning was localized to the weight-bearing tibial-femoral contact point, which is the point of the greatest weight bearing within the knee. Thus loss of cartilage was greatest where the loading is the generally the greatest -- reducing pressure across the knee during flight had the biggest negative impact at that location. Within the knee cartilage, enzymes capable of breaking down cartilage (MMPs) were increased in the spaceflight FLIGHT cartilage. Additionally, the molecules that provide the spongy, compressive properties of cartilage were lower after spaceflight. The meniscus is a tissue in the knee that stabilizes the structure. We found that Meniscal volume was significantly lower than controls after flight. Additionally, the same molecules (proteoglycans) that provide the compressive properties in cartilage were also lower in the menisci of FLIGHT mice. This pattern was similar to results from the STS-135 space shuttle flight flight, in which proteoglycans were lower in menisci from left (-11%; p<0.05) and right (-9%; p=.1) knees vs controls. We also found that within the menisci, the "control pathways" that can lead to damage or arthritis were activated, specifically ones that can result in a decrease in meniscal volume. Many proteins associated with building new cartilage and meniscal tissues were lower after flight, and this could be due to lowered defenses against free radicals.

SUMMARY: Degradation of knee soft tissues occurred after flights on ISS. Cartilage loss localized to the highest weight-bearing location across the knees. Excess oxidative stress is a known cause for meniscal and cartilage degradation; our data indicate this as potentially a contributor to the observed joint degradation.

B2. Gait Assessment Findings. We have submitted our gait For FLIGHT mice; many of the evaluated gait characteristics in the hind limbs were significantly changed, including: stride width variability; stride length and variance; stride, swing, and stance duration; paw angle and area at peak stance; and step angle, among others. Gait characteristics that decreased included stride frequency, and others. Moreover, numerous forelimb gait characteristics in the FLIGHT mice were changed at post-flight measures relative to pre-flight. We also sought to identify gait alterations that are reflective of joint (cartilage) degradation, a known response of knees to reduced weight bearing. Some patterns of gait change in rodents reflect musculoskeletal deficits, a well-described consequence of prolonged periods of reduced weight bearing in mice. For instance, an arthritic gait in rodents may display increased stride duration and reduced stride length and/or increased stride width, with related pain. Increased stride duration was observed in the flight animals, but as noted the stride length was significantly increased and stride width was unaltered, which is in contrast to what one might expect with painful arthritis. Thus while cartilage degradation was noted from anatomic and proteomic assessment, the reponse may not be painful at the point of tissue harvest.

B3. Muscle Metabolomic findings. We examined how metabolism was changed in the gastrocnemius, and the quadriceps femoris (“quadriceps”) muscles, and was conducted at Georgetown University by Dr. Willey’s collaborator, Dr. Lia Laiakis. Results demonstrate the increased responsiveness of gastrocnemius to spaceflight compared to quadricep in the metabolomic level. All the data suggest that microgravity and spaceflight have a direct effect on the metabolomic profiles of muscle tissues. However, some muscles appear to be more responsive compared to others, or some have the ability to reprogram efficiently upon return to Earth.

NOTE: End date changed to 3/31/2020 per F. Hernandez/ARC (Ed., 6/23/17)

To study these problems, we will determine the hip and knee joint damage that occurs in mice that will fly in space on the International Space Station for 30 days. This damage will be compared to the hip and knee joint damage in another group of mice kept on Earth that also will not have weight on the hip and knee joints for 30 days, with or without receiving radiation exposure that simulates a solar flare. Damage to the hip and knee joint structures will be determined using imaging techniques, engineering devices to measure tissue strength, stained tissue sections, and identification of the molecules that cause the damage. The ability to walk normally after 30 days of weightlessness will also be determined. Finally, we will determine if treadmill running or climbing can reverse any of the hip and knee joint damage caused by being in the weightless space environment.

Our goal is to determine, 1] if hip and knee joint damage occurs in the weightless space environment, and 2] if recovery from this damage is possible with exercise.

Primary Results: Contrast-enhanced microCT demonstrated decreased in volume and thickness at the site of primary weight-bearing in the knee (e.g., the femoral-tibial cartilage-cartilage contact point) in all treatment groups vs. GROUND-SHAM. Interestingly and importantly, damage was localized to the sites of most weight bearing; cartilage is senstiive to loading. Within the knee cartilage, the protein collagen was reduced, and collagen type II-degrading matrix metalloproteinase-13 (MMP-13) were greater in all groups vs. controls indicating that cartilage degraded after both radiation and reduced weight bearing. Circulating serum cartilage oligomeric matrix proteins (sCOMP), biomarker for ongoing cartilage degradation, was increased in all of the irradiated groups vs. GROUND-SHAM, regardless of unloading. Mass spectrometry, combined with Ingenuity Pathway Analysis (IPA), of the cartilage lining the femoral head showed decrease in cartilage compositional proteins, increased osteoarthritic pathways, and decreased cell survival by increased apoptosis and oxidative stress. Our findings demonstrate that both individually and combined HLU and spaceflight-relevant doses of irradiation lead to cartilage degradation of the knee and hip with expression of an arthritic phenotype. Moreover, early administration of low dose irradiation (0.1 Gy, 0.5 Gy, or 1 Gy) causes an active catabolic response in cartilage 24 days post irradiation. IMp

2. Flight study: Our mouse payload has been launched aboard the SpaceX-12 mission to the International Space Station. We successfully brought our Digigait treadmill down to the Kennedy Space Center, performed an initial gait assessment on all mice; grouped the mice; and after launch collected the baseline tissues for ourselves and for biospecimen sharing program (BSP). We traveled to Loma Linda University, where we repeated the gait assessment on the flight animals upon return, a new Cohort Control group of mice, and again collected tissues for our primary science and for BSP. We have analyzed how gait is altered post flight. Analysis of tissues is ongoing.

Primary results: Gait data were acquired over ~1.5 minutes/mouse. Several patterns of gait changes were noted post-flight. Stride width variance increased in both forelimbs (p<0.05) and hind limbs (p<0.001), indicating an increased risk of falling. Increased swing phase duration observed in the hind limbs post-flight (p < 0.05) characterizes an arthritic gait. Step angle widened in hind limbs post-flight, suggesting impaired CNS motor centers with ataxia (p < 0.01). Paw area variability at peak stance increased in hind limbs (p < 0.01), reflecting impaired neuromotor control. Importantly, while many gait patterns indicated neuromotor deficits, most remained unaltered. Our data indicates that longitudinal gait analysis of rodents provides a non-invasive assay to measure functional responses to spaceflight. The mobile DigiGait system permits identification of gait changes in rodents suggestive of functional impairment. The majority of deficits from RR-9 mice were noted as sensorimotor in nature. Deficits were more pronounced in hind limbs vs. forelimbs, which could be related to locomotor habits in the Rodent Habitats on ISS (e.g., grabbing wire mesh).

NOTE: End date changed to 3/31/2020 per F. Hernandez/ARC (Ed., 6/23/17)

To study these problems, we will determine the hip and knee joint damage that occurs in mice that will fly in space on the International Space Station for 30 days. This damage will be compared to the hip and knee joint damage in another group of mice kept on Earth that also will not have weight on the hip and knee joints for 30 days, with or without receiving radiation exposure that simulates a solar flare. Damage to the hip and knee joint structures will be determined using imaging techniques, engineering devices to measure tissue strength, stained tissue sections, and identification of the molecules that cause the damage. The ability to walk normally after 30 days of weightlessness will also be determined. Finally, we will determine if treadmill running or climbing can reverse any of the hip and knee joint damage caused by being in the weightless space environment.

Our goal is to determine, 1] if hip and knee joint damage occurs in the weightless space environment, and 2] if recovery from this damage is possible with exercise.

Specific progress includes:

1. We finished all the tail suspension +/- simulated spaceflight radiation studies. Mice were either hindlimb unloaded (HLU) (n=40) or remained full weight-bearing as Ground mice (n=40). Within each of these groups, mice either received no radiation, or 1 of 3 low-dose, spaceflight-relevant radiation scenarios. We measured cartilage and joint damage on Day 30 after initiating HLU.

3. From our ground-based studies, we noted from our advanced imaging analysis that both reduced weight bearing and low dose radiation can cause loss of cartilage and molecular changes characteristic of arthritis. The volume and thickness of cartilage lining the medial tibial plateau (knee cartilage) was lower in the HLU groups than the GROUND mice whether or not the mice were irradiated. There was not a combined effect; therefore, both reduced weight bearing and radiation can cause loss of cartilage volume. Also, we noted an increase in the presence of enzymes and molecules that are associated with a breakdown of cartilage in the hip cartilage after HLU and irradiation. A specific change in two cell signaling pathways was observed in the femoral head cartilage that is strongly associated with cartilage catabolism and arthritis. Inflammation may have been increased in the HLU mice as indicated by serum IL-6 levels; increased IL-6 is associated with arthritis. Finally, there was an overall reduction in the amount of normal proteins that make up the femoral head cartilages. Taken together, both HLU and radiation can damage cartilage and joints.

4. In preparation for our flight study, we performed a rodent study in which we compared the walking gait of mice before and after 30 days of reduced weight bearing via tail suspension (HLU) with those that were not unloaded (GROUND). We utilized our Digigait system to measure the walking gait. Analysis has not been performed as of yet, as we began preparation for our flight investigation.

5. Flight study: Our mouse payload has been launched aboard the SpaceX-12 mission to the International Space Station, and is currently in orbit. We measured the walking gait of the mice before launch. We plan to recover the mice in September 2017.

To study these problems, we will determine the hip and knee joint damage that occurs in mice that will fly in space on the International Space Station for 30 days. This joint damage will be compared to the hip and knee joint damage in another group of mice kept on Earth that also will not have weight on the hip and knee joints for 30 days, with or without receiving radiation exposure that simulates a solar flare. Damage to the hip and knee joint structures will be determined using imaging techniques, engineering devices to measure tissue strength, stained tissue sections, and identification of the molecules that cause the damage. The ability to walk normally after 30 days of weightlessness will also be determined. Finally, we will determine if treadmill running or climbing can reverse any of the hip and knee joint damage caused by being in the weightless space environment.

Our goal is to determine, 1] if hip and knee joint damage occurs in the weightless space environment, and 2] if recovery from this damage is possible with exercise.

A. We developed a chamber that lets us simulate the spaceflight and near weightless radiation environment at the same time. We can place mice into this this chamber and expose them to a similar dose of radiation that astronauts can face while in orbit, all while the mice are exposed to reduced weight bearing. This setup can be used at nearly all academic medical centers. These animals do not require anesthesia during the procedure. Thus this chamber improves and expands our ability to perform ground-based research that simulates the challenges of the spaceflight environment

B. We have identified that periods of reduced weight-bearing causing thinning of the cartilage lining the tibial plateau. Using resources that were allocated prior to full implementation, we developed an ultra-high resolution nano-computed tomography (nanoCT) method of measuring cartilage changes after periods of hindlimb unloading (HLU). The medial plateau in particular is important as this is the region of the knee with the highest amount of weight-bearing, and is often the region most susceptible to arthritis.

D. Indicators of arthritis are increased within the femoral head cartilage after periods of reduced weight-bearing. Specifically, the genes that drive degradation of cartilage during the process of arthritis are increased after 21 days of HLU. The pattern of altered gene expression in the femoral heads after 21 days of HLU are highly indicative of a specific type of intracellular signaling (Wnt-signaling) in cartilage that contributes to cartilage damage and arthritis.

To study these problems, we will determine the hip and knee joint damage that occurs in mice that will fly in space on the International Space Station for 30 days. This joint damage will be compared to the hip and knee joint damage in another group of mice kept on Earth that also will not have weight on the hip and knee joints for 30 days. Damage to the hip and knee joint structures will be determined using imaging techniques, engineering devices to measure tissue strength, stained tissue sections, and identification of the molecules that cause the damage. The ability to walk normally after 30 days of weightlessness will also be determined. Finally, we will determine if treadmill running or climbing can reverse any of the hip and knee joint damage caused by being in the weightless space environment.

Our goal is to determine, 1] if hip and knee joint damage occurs in the weightless space environment, and 2] if recovery from this damage is possible with exercise. From these studies, we also will gain insights into how arthritis develops in wheel-chair bound spinal cord injury patients or after limb surgery, and how it can be prevented.

To study these problems, we will determine the hip and knee joint damage that occurs in mice that will fly in space on the International Space Station for 30 days. This joint damage will be compared to the hip and knee joint damage in another group of mice kept on Earth that also will not have weight on the hip and knee joints for 30 days. Damage to the hip and knee joint structures will be determined using imaging techniques, engineering devices to measure tissue strength, stained tissue sections, and identification of the molecules that cause the damage. The ability to walk normally after 30 days of weightlessness will also be determined. Finally, we will determine if treadmill running or climbing can reverse any of the hip and knee joint damage caused by being in the weightless space environment.

Our goal is to determine, 1] if hip and knee joint damage occurs in the weightless space environment, and 2] if recovery from this damage is possible with exercise. From these studies, we also will gain insights into how arthritis develops in wheel-chair bound spinal cord injury patients or after limb surgery, and how it can be prevented.