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Project Title:  Spinal Structure and Function after 90 Days Long-Duration Simulated Space Flight and Recovery Reduce
Fiscal Year: FY 2019 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 08/01/2014  
End Date: 12/31/2018  
Task Last Updated: 03/23/2019 
Download report in PDF pdf
Principal Investigator/Affiliation:   Hargens, Alan R. Ph.D. / University of California, San Diego 
Address:  Altman Clinical and Translational Research Institute 
9452 Medical Center Drive/0863 
La Jolla , CA 92037-0863 
Email: ahargens@ucsd.edu 
Phone: 858-534-7837  
Congressional District: 52 
Web:  
Organization Type: UNIVERSITY 
Organization Name: University of California, San Diego 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Ferguson, Adam  Ph.D. University of California at San Francisco 
Lotz, Jeffrey  Ph.D. University of California at San Francisco 
Macias, Brandon  Ph.D. NASA Johnson Space Center 
Masuda, Koichi  M.D. University of California at San Diego 
Project Information: Grant/Contract No. NNX14AP25G 
Responsible Center: NASA JSC 
Grant Monitor: Norsk, Peter  
Center Contact:  
Peter.norsk@nasa.gov 
Solicitation / Funding Source: 2013 HERO NNJ13ZSA002N-Crew Health (FLAGSHIP & NSBRI) 
Grant/Contract No.: NNX14AP25G 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Human Research Program Elements: (1) HHC:Human Health Countermeasures
Human Research Program Risks: (1) Osteo:Risk Of Early Onset Osteoporosis Due To Spaceflight (No longer used, July 2020)
Human Research Program Gaps: (1) Osteo04:We do not know the contribution of each risk factor on bone loss and recovery of bone strength, and which factors are the best targets for countermeasure application (IRP Rev E)
Flight Assignment/Project Notes: NOTE: End datd changed to 12/31/2018 per NSSC information (Ed., 7/18/17)

Task Description: The vertebral bodies and flexible intervertebral discs are important, weight-bearing tissues that have adapted to gravitational stress. Consequently, the absence of gravitational axial loads during exposure to microgravity likely disrupts normal spine physiology. Throughout longer space flight missions, deconditioning of the intervertebral discs and spinal muscles poses a serious injury risk upon re-exposure to upright posture in a gravitational environment. We will use state-of-the-art technologies to quantify morphology, biochemistry, and kinematics of spines (including the vertebrae, intervertebral discs, and spinal muscles) of rats at defined time points as described in the NASA research announcement. After successful completion of our investigation, we will deliver a comprehensive database of simulated microgravity-induced spinal adaptations (type and magnitude). The overarching goal of these proposed studies is to develop a long-duration space flight ground based model of spine function and structure. In addition, this research project will afford the opportunity to examine possible gender differences in spinal structure and function. Our research group is in a unique position to leverage our past rodent space flight experience on STS-131, STS-133, STS-135, and BION M-1 missions and directly compare to this ground based model of simulated microgravity. Moreover, we are also uniquely positioned to compare this 90-days hindlimb unloading model to those changes that occur in our currently funded project to test crew members before and after 6-month International Space Station (ISS) missions. Our project directly addresses Critical Path Roadmap Risks and Questions regarding disc injury (Integrated Research Plan (IRP) Gap-B4)): Is damage to joint structure, intervertebral discs, or ligaments incurred during or following microgravity exposure? Our research will improve understanding of the underlying pathophysiology of spinal deconditioning induced by simulated microgravity, and mechanisms of spinal adaptation following re-exposure to 1-G. Our long-term goal is to prevent such spinal deconditioning with exercise or other physiologic countermeasures. The goal of this research is to comprehensively characterize 90-days simulated space flight and recovery induced changes in spinal tissue morphology, biochemistry, and biomechanics.

Research Impact/Earth Benefits: To our knowledge, this study is the first to examine the effects of 90-days simulated space flight on spinal deconditioning in rats and to compare this model of simulated microgravity with actual space flight. The vertebral bodies and flexible intervertebral discs are important, weight-bearing tissues that have adapted to gravitational stress. Our research will aid understanding of spinal deconditioning during simulated microgravity and of the higher incidence of disc prolapse or herniation following re-exposure to 1-G with a long-term view to prevent such spinal deconditioning with exercise or other physiologic countermeasures. This research may aid understanding of spinal deconditioning during inactivity such as after spinal cord injury and bed rest in human patients on Earth.

Task Progress & Bibliography Information FY2019 
Task Progress: INTRODUCTION

The vertebral bodies and flexible intervertebral discs are important, weight-bearing tissues that have adapted to gravitational stress. Consequently, the absence of gravitational axial loads during exposure to microgravity likely disrupts normal spine physiology. Throughout longer space flight missions, deconditioning of the intervertebral discs and spinal muscles poses a serious injury risk upon re-exposure to upright posture in a gravitational environment. Hind-limb suspension of rats is accepted as a ground-based model of microgravity. This model involves elevating rodents by the tail to produce a head-down tilt of approximately 30 degrees, effectively unloading the hind limbs. This unloading model reproduces some physiological adaptations to spaceflight such as hind-limb bone and muscle loss, but it’s unclear if this model is appropriate for studies of intervertebral discs. However, this rodent model provides an invasive way to test spinal biomechanics, not possible in humans exposed to actual or simulated microgravity. We have previously demonstrated that the caudal (tail) discs from mice exposed to 15 days of space flight had reduced bending strength and range of motion, as well as difference in failure sites using four-point bending tests. Control segments fail near the bone-disc endplate junction, whereas the space flight segments fail in the bone at the growth plate. In this study, we hypothesized that caudal motion segments subjected to hind-limb suspension would behave similarly to those from space flight rodents when compared to controls.

Our research group was in a unique position to leverage our past rodent space flight experience on STS-131, STS-133, STS-135, and BION M-1 missions and directly compare to this ground-based model of simulated microgravity. Moreover, we were also uniquely positioned to compare this 90-days hind-limb suspension model to those changes that occur in our currently funded project to test crew members before and after 6-month International Space Station (ISS) missions. Our project directly addresses Critical Path Roadmap Risks and Questions regarding disc injury (IRP Gap-B4): Is damage to joint structure, intervertebral discs, or ligaments incurred during or following microgravity exposure? Our research will improve understanding of the underlying pathophysiology of spinal deconditioning induced by simulated microgravity, and mechanisms of spinal adaptation following re-exposure to 1-G. Our long-term goal is to prevent such spinal deconditioning with exercise or other physiologic countermeasures. The goal of this research is to characterize 90-days simulated space flight and recovery induced changes comprehensively in spinal tissue morphology, biochemistry, and biomechanics.

METHODS

To investigate the effect of hind-limb suspension, we analyzed the spines and discs of many groups of rats, comparing them to controls and previous flight mice on STS-131. Our joint University of California at San Diego-niversity of California at San Francisco (UCSD-UCSF) team made significant progress to integrate and implement tissue sharing procedures as part of our NASA award (NNX14AP25G).

Initially, key team members, Dr. Brandon Macias of UCSD and Dr. Kazuhito Morioka of UCSF traveled to UC-Davis to train and implement rodent calvaria, spine, and tail tissue dissection/collection and storage procedures. Due to the reassignment of the trained NASA dissection team to NASA-Kennedy Space Center, additional training and travel to UC-Davis were required to train new NASA personnel at the UC-Davis site. Our team held an in-service at UCSD to finalize spinal cord expulsion procedures and to verify that this expulsion procedure would not damage vertebral and disc structures. These practice spinal cord procedures, practice tissues dissection, and freezing procedures were successful. Following successful verification of our procedures within out UCSD and UCSF team we worked to integrate and implement them at the UC-Davis site. In addition, our team reviewed and developed a tissue sharing plan within our UCSD/UCSF team. Tissues were centralized at UCSF. Following retrieval of the spinal cord tissue at UCSF, spines were shipped to UCSD for micro-computed tomography analysis of the vertebral bodies. Following completion of micro-computed tomography on tissues from UC-Davis, they were shipped to UCSF for intervertebral disc analysis. Overall this project required timely communication, coordination, and logistical planning to ensure high-quality tissue processing and research results.

Vertebral Bone Studies

Thirty-seven Long Evans rats (3 months old) were separated into two groups, hindlimb-unloaded (HLU) group and weight-bearing control (WBC) group. Rats in the HLU group were subjected to hindlimb suspension, as reported by Morey-Holton and Globus, for 14 days (n=7, 14D), 90 days (n=8, 90D), and 90 days with 28 days of recovery (n=3, 90D/Recov). The hindlimb suspension was removed during the 28-day recovery period. The WBC group was not subjected to the suspension but was similarly divided into groups of 14D (n=7), 90D (n=9), and 90D/Recov (n=3). NASA-defined protocols were followed in the hindlimb unloading of the rats. After each time point, the groups were sacrificed, and lumbar spines were harvested.

The spines (Th12-S1) were scanned in a Skyscan 1076 Micro-CT scanner (Bruker, Kontich, Belgium) at 9 µm resolution (70kV, 141µA) with a 1.0 mm aluminum filter and hydroxyapatite phantom rods (4mm diameter, 0.25 and 0.75 g/cm3) for BMD (bone mineral density) analysis. Data viewer and CT-Analyzer software (Bruker, Kontich, Belgium) were used to analyze the spines for BMD (g/cm3) and bone morphology parameters, such as percent bone volume (bone volume/tissue volume, BV/TV), trabecular thickness (Tb.Th, mm), and trabecular separation (Tb.Sp, mm). To analyze IVD height, a custom written C++ program calculated the mean disc height distance (DHD) from vertebral endplates isolated in Mimics (Materialize, Leuven, Belgium). All data were analyzed using three or two-way ANOVA with Fisher PLSD post hoc tests (p<0.05). Level, group, and time point were treated as independent factors.

RESULTS

On average, intervertebral discs in control rats had a normalized strength of 0.49 ± 0.16 N/mm2 and unloaded had a value of 0.52 ± 0.16 N/mm2 (p>0.05). The stiffness and toe displacement of controls were 16.5 ± 6.87 N/mm and 0.67 ± 0.19 mm, respectively, while the unloaded group had a stiffness of 17.7 ± 6.88 N/mm and toe displacement of 0.66 ± 0.19 mm (p>0.05). Histological analyses showed consistent failure among all groups at the endplate junction, where the annulus fibers attach to the fibrocartilage of the bony endplate.

Vertebral Bone Results: There were no significant changes among all weight-bearing control (WBC) groups for all levels, groups, and time points.

Bone Mineral Density (BMD) analysis: Group and time significantly affected BMD. BMD of the 14D hindlimb-unloaded (HLU) and 90D HLU groups were significantly lower than those of the 14D WBC and 90D WBC groups, respectively (both, p<0.05). BMD of the 90D group was significantly lower than that of the 14D HLU group (p<0.01); however, BMD of the 90D/Recov HLU group was significantly higher than that of the 90D HLU and 14D HLU groups (p<0.01 for 90D HLU, p<0.05 for 14D HLU).

Bone morphological analysis: Similarly, group and time significantly affected BV/TV, Tr. Th, and Tr. Sp. The 14D HLU and the 90D HLU groups had a significantly lower BV/TV than those of corresponding WBC groups (vs. 14D WBC, vs. 90D WBC, respectively, both p<0.01). There was no significant progression of BT/TV decrease between the 14D HLU and 90D HLU groups after the initial decrease; importantly, BV/TV of the 90D/Recov HLU group was significantly greater than that of 90D HLU group (p<0.01). Tr. Th of the 14D HLU group was significantly lower than that of the 14D WBC and 90D HLU groups (p<0.01, respectively). The 90D/Recov HLU group had a significantly higher Tr. Th those of the 14D HLU, 90D HLU, and 90D/Recov WBC groups (p<0.01 for all). For Tr. Sp, the 90D HLU group had a significantly higher separation than that of 90D WBC group (p<0.01). Tr. Sp of the 90D HLU group was significantly greater than of the 14D HLU group (p<0.01); no significant difference was observed between the 90D HLU and the 90D/Recov HLU groups.

Disc height distance (DHD) analysis: Group and time significantly affected DHD. DHD of the 90D HLU group was significantly lower than that of the 90D WBC group (p<0.01). DHD differences were not significant in the HLU and WBC groups of the 14D and 90D/Recov time point. The 14D HLU DHD was a significantly higher than that of the 90D HLU and 90D/Recov groups (p<0.01 for both). Importantly, there were no significant differences in DHD between the 90D HLU and the 90D/Recov HLU groups.

DISCUSSION

There were no statistically significant differences in biomechanical values or failure mode between control and hind-limb suspended groups. These results suggest that hind-limb suspension may not be a useful model for detecting the effects of unloading on caudal (tail) intervertebral discs of rats. Previous studies of the hind-limb suspension model in our lab document that approximately 50% of the rat’s body weight is taken up by tension on the tail. This is not the case in actual microgravity where the intervertebral discs are unloaded except during muscle contractions that are a mild non-gravitation mode of disc loading.

Bone Mineral Density (BMD) analyses of rat vertebral bodies document a significant decrease in the 90D hindlimb-unloaded (HLU) group compared to the 14D HLU group and a significant recovery in BMD in the 90D/Recov group compared to the 90D HLU group. The initial decrease in BMD agrees with previous studies that reveal a similar decrease in mice sent on a 15-day space mission. BMD and bone morphological parameters generally show a significant difference between the 90D HLU and the 90D/Recov HLU groups. However, there are no significant differences between the HLU and WBC groups of the 90D/Recov time point in BMD, BV/TV, Tr. Th, and Tr. Some results suggest that a recovery period can aid in bone recovery to baseline conditions. To examine the extent of bone recovery after hindlimb suspension, future analyses of the bone recovery at different time points is needed.

SIGNIFICANCE

Extended unloading of the hindlimb decreases BMD, bone morphological parameters, and progressive decrease of intervertebral discs height. The 28-day recovery period, where the hindlimb suspension is removed, aids in bone recovery but is ineffective in intervertebral discs height restoration. To our knowledge, this study is the first to examine the effects of 90-days simulated space flight on spinal deconditioning in rats and to compare this model of simulated microgravity with mice exposed to actual space flight. Thus, the vertebral bodies and flexible intervertebral discs are important, weight-bearing tissues that adapt to gravitational stress.

RESEARCH IMPACT/EARTH BENEFIT

Our research will aid understanding of spinal deconditioning during simulated microgravity and of the higher incidence of disc prolapse or herniation following re-exposure to 1-G with a long-term view to prevent such spinal deconditioning with exercise or other physiologic countermeasures. This research may aid understanding of spinal deconditioning during inactivity such as after spinal cord injury and bed rest in human patients on Earth.

ACKNOWLEDGMENTS: We thank the Animal Resources Center at the University of California, Davis and the Space Biosciences Division at NASA Ames Research Center.

Bibliography Type: Description: (Last Updated: 12/08/2021)  Show Cumulative Bibliography Listing
 
Articles in Peer-reviewed Journals Howden M, Siamwala JH, Hargens AR. "Bone microvascular flow differs from skin microvascular flow in response to head-down tilt." J Appl Physiol (1985). 2017 Oct 1;123(4):860-6. Epub 2017 Jun 29. https://doi.org/10.1152/japplphysiol.00881.2016 ; PubMed PMID: 28663380 , Oct-2017
Articles in Peer-reviewed Journals Vico L, Hargens A. "Skeletal changes during and after spaceflight." Nat Rev Rheumatol. 2018 Mar 21;14(4):229-45. Review. https://doi.org/10.1038/nrrheum.2018.37 ; PubMed PMID: 29559713 , Mar-2018
Articles in Peer-reviewed Journals Zhang LF, Hargens AR. "Spaceflight-induced intracranial hypertension and visual impairment: Pathophysiology and countermeasures." Physiol Rev. 2018 Jan 1;98(1):59-87. Review. https://doi.org/10.1152/physrev.00017.2016 ; PubMed PMID: 29167331 , Jan-2018
Articles in Peer-reviewed Journals Pandiarajan M, Hargens AR. "Ground-based analogs for human spaceflight." Front Physiol. 2020 Jun 23;11:716. https://doi.org/10.3389/fphys.2020.00716 ; PMID: 32655420 ; PMCID: PMC7324748 , Jun-2020
Awards Snyder A. "Medical student mentee, American Physiological Society Exercise & Environmental Physiology Section’s National Space Biomedical Research Institute’s Gravitational Physiology Predoctoral Award, Experimental Biology, Boston, MA, April 2015." Apr-2015
Awards Siamwala J. "Postdoctoral mentee. American Physiological Society Exercise & Environmental Physiology Section’s National Space Biomedical Research Institute Gravitational Physiology Beginning Investigator Award for abstract, Siamwala JH, BR Macias, R Healey, and AR Hargens. 'Cerebral Vascular Changes in Space Mice Calvaria.' Experimental Biology, Boston, MA, April 2015." Apr-2015
Awards Khieu K. "Kristine Khieu, a UCSD senior in Bioengineering, received the 2017 USRA Frederick A. Tarantino Memorial Scholarship Award. She was selected from among 112 eligible applicants for one of 6 USRA scholarships. October 2017." Oct-2017
Awards Hargens A. "Citation Award from American College of Sports Medicine, May 2015." May-2015
Awards Hargens A. "Kjell Johansen Award and Invited Lecture “What can Giraffes on Earth Teach Astronauts in Space?” University of Aarhus, Denmark, April 2016." Apr-2016
Awards Hargens A. "Recognition Award from Southest American College of Sports Medicine, October 2017." Oct-2017
Awards Hargens A. [Alan Hargens] "NASA Distinguished Public Service Metal (NASA’s highest form of recognition that is awarded to any non-Government individual or to an individual who was not a Government employee during the period in which the service was performed, whose distinguished service, ability, or vision has personally contributed to NASA's advancement of United States' interests. The individual's achievement or contribution must demonstrate a level of excellence that has made a profound or indelible impact to NASA mission success; therefore, the contribution is so extraordinary that other forms of recognition by NASA would be inadequate). June 2017 " Jun-2017
Project Title:  Spinal Structure and Function after 90 Days Long-Duration Simulated Space Flight and Recovery Reduce
Fiscal Year: FY 2017 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 08/01/2014  
End Date: 12/31/2018  
Task Last Updated: 07/03/2017 
Download report in PDF pdf
Principal Investigator/Affiliation:   Hargens, Alan R. Ph.D. / University of California, San Diego 
Address:  Altman Clinical and Translational Research Institute 
9452 Medical Center Drive/0863 
La Jolla , CA 92037-0863 
Email: ahargens@ucsd.edu 
Phone: 858-534-7837  
Congressional District: 52 
Web:  
Organization Type: UNIVERSITY 
Organization Name: University of California, San Diego 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Ferguson, Adam  Ph.D. University of California at San Francisco 
Lotz, Jeffrey  Ph.D. University of California at San Francisco 
Macias, Brandon  Ph.D. NASA Johnson Space Center 
Masuda, Koichi  M.D. University of California at San Diego 
Project Information: Grant/Contract No. NNX14AP25G 
Responsible Center: NASA JSC 
Grant Monitor: Norsk, Peter  
Center Contact:  
Peter.norsk@nasa.gov 
Solicitation / Funding Source: 2013 HERO NNJ13ZSA002N-Crew Health (FLAGSHIP & NSBRI) 
Grant/Contract No.: NNX14AP25G 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:  
No. of Bachelor's Candidates:
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:
Human Research Program Elements: (1) HHC:Human Health Countermeasures
Human Research Program Risks: (1) Osteo:Risk Of Early Onset Osteoporosis Due To Spaceflight (No longer used, July 2020)
Human Research Program Gaps: (1) Osteo04:We do not know the contribution of each risk factor on bone loss and recovery of bone strength, and which factors are the best targets for countermeasure application (IRP Rev E)
Flight Assignment/Project Notes: NOTE: End datd changed to 12/31/2018 per NSSC information (Ed., 7/18/17)

Task Description: The vertebral bodies and flexible intervertebral discs are important, weight-bearing tissues that have adapted to gravitational stress. Consequently, the absence of gravitational axial loads during exposure to microgravity likely disrupts normal spine physiology. Throughout longer space flight missions, deconditioning of the intervertebral discs and spinal muscles poses a serious injury risk upon re-exposure to upright posture in a gravitational environment. We will use state-of-the-art technologies to quantify morphology, biochemistry, and kinematics of spines (including the vertebrae, intervertebral discs, and spinal muscles) of rats at defined time points as described in the NASA research announcement. After successful completion of our investigation, we will deliver a comprehensive database of simulated microgravity-induced spinal adaptations (type and magnitude). The overarching goal of these proposed studies are to develop a long-duration space flight ground based model of spine function and structure. In addition, this research project will afford the opportunity to examine possible gender differences in spinal structure and function. Our research group is in a unique position to leverage our past rodent space flight experience on STS-131, STS-133, STS-135, and BION M-1 missions and directly compare to this ground based model of simulated microgravity. Moreover, we are also uniquely positioned to compare this 90-days hindlimb suspension model to those changes that occur in our currently funded project to test crew members before and after 6-month International Space Station (ISS) missions. Our project directly addresses Critical Path Roadmap Risks and Questions regarding disc injury (Integrated Research Plan (IRP) Gap-B4)): Is damage to joint structure, intervertebral discs, or ligaments incurred during or following microgravity exposure? Our research will improve understanding of the underlying pathophysiology of spinal deconditioning induced by simulated microgravity, and mechanisms of spinal adaptation following re-exposure to 1-G. Our long-term goal is to prevent such spinal deconditioning with exercise or other physiologic countermeasures. The goal of this research is to comprehensively characterize 90-days simulated space flight and recovery induced changes in spinal tissue morphology, biochemistry, and biomechanics.

Research Impact/Earth Benefits: To our knowledge, this study is the first to examine the effects of 90-days simulated space flight on spinal deconditioning in rats and to compare this model of simulated microgravity with actual space flight. The vertebral bodies and flexible intervertebral discs are important, weight-bearing tissues that have adapted to gravitational stress. Our research will aid understanding of spinal deconditioning during simulated microgravity and of the higher incidence of disc prolapse or herniation following re-exposure to 1-G with a long-term view to prevent such spinal deconditioning with exercise or other physiologic countermeasures. This research may aid understanding of spinal deconditioning during inactivity such as after spinal cord injury and bed rest in human patients on Earth.

Task Progress & Bibliography Information FY2017 
Task Progress: Introduction: Astronauts are at increased risk for spinal fractures and disc herniation because of the lack of loading during space missions, which can cause bone mineral density (BMD) decline and micro-architectural changes in structural integrity [1-3]. It may take several years to achieve complete BMD recovery [4]. The underlying mechanism and magnitude of BMD, bone morphology and intervertebral disc (IVD) changes remain unclear [5]. To study these effects, the hindlimb unloading model, a ground-based model that mimics cephalic fluid shifts and bone volume density losses of a microgravity environment [6-7], was utilized because of the limited pool of human subjects and the resource restrictions that a space flight animal study would require.

In this model, we hypothesized that lumbar spines would experience a significantly greater decline in BMD, bone morphological parameters, and IVD height with longer exposure to hindlimb unloading and that these changes are restorable with a recovery period. A 90-day hindlimb unloading study previously reported a loss in BMD and IVD height [8]. However, there are no data that encompass multiple time points or investigate the extent of recovery under the normal gravity conditions after unloading. The purpose of this study was to investigate the progression of changes in BMD and IVD during unloading and the extent of recovery after returning to normal gravity conditions by quantifying BMD, bone morphology, and IVD height at different time points.

Methods: Thirty-seven Long Evans rats (3 months old) were separated into two groups, hindlimb-unloaded (HLU) group and weight-bearing control (WBC) group. Rats in the HLU group were subjected to hindlimb unloading, as reported by Morey-Holton and Globus, for 14 days (n=7, 14D), 90 days (n=8, 90D), and 90 days with 28 days of recovery (n=3, 90D/Recov) [6]. The hindlimb unloading was removed during the 28-day recovery period. The WBC group was not subjected to the unloading but was similarly divided into groups of 14D (n=7), 90D (n=9), and 90D/Recov (n=3). NASA-defined protocols were followed in the hindlimb unloading of the rats. After each time point, the groups were sacrificed, and lumbar spines were harvested.

The spines (Th12-S1) were scanned in a Skyscan 1076 Micro-CT scanner (Bruker, Kontich, Belgium) at 9 µm resolution (70kV, 141µA) with a 1.0 mm aluminum filter and hydroxyapatite phantom rods (4 mm diameter, 0.25 and 0.75 g/cm3) for BMD analysis. Dataviewer and CT-Analyser software (Bruker, Kontich, Belgium) were used to analyze the spines for BMD (g/cm3) and bone morphology parameters, such as percent bone volume (bone volume/tissue volume, BV/TV), trabecular thickness (Tb.Th, mm), and trabecular separation (Tb.Sp, mm) [9-10]. To analyze IVD height, a custom written C++ program calculated the mean disc height distance (DHD) from vertebral endplates isolated in Mimics (Materialize, Leuven, Belgium) [11]. All data were analyzed using three or two-way ANOVA with Fisher PLSD post hoc tests (p<0.05). Level, group, and time point were treated as independent factors.

Results: There were no significant changes among all WBC groups for all levels, groups, and time points.

BMD analysis: Group and time significantly affected BMD. BMD of the 14D HLU and 90D HLU groups were significantly lower than those of the 14D WBC and 90D WBC groups, respectively (both, p<0.05). BMD of the 90D group was significantly lower than that of the 14D HLU group (p<0.01); however, BMD of the 90D/Recov HLU group was significantly higher than that of the 90D HLU and 14D HLU groups (p<0.01 for 90D HLU, p<0.05 for 14D HLU).

Bone morphological analysis: Similarly, group and time significantly affected BV/TV, Tr. Th, and Tr. Sp. The 14D HLU and the 90D HLU groups had a significantly lower BV/TV than those of corresponding WBC groups (vs. 14D WBC, vs. 90D WBC, respectively, both p<0.01). There was no significant progression of BT/TV decrease between the 14D HLU and 90D HLU groups after the initial decrease; importantly, BV/TV of the 90D/Recov HLU group was significantly greater than that of 90D HLU group (p<0.01). Tr. Th of the 14D HLU group was significantly lower than that of the 14D WBC and 90D HLU groups (p<0.01, respectively). The 90D/Recov HLU group had a significantly higher Tr. Th those of the 14D HLU, 90D HLU, and 90D/Recov WBC groups (p<0.01 for all). For Tr. Sp, the 90D HLU group had a significantly higher separation than that of 90D WBC group (p<0.01). Tr. Sp of the 90D HLU group was significantly greater than of the 14D HLU group (p<0.01); no significant difference was observed between the 90D HLU and the 90D/Recov HLU groups.

DHD analysis: Group and time significantly affected DHD. DHD of the 90D HLU group was significantly lower than that of the 90D WBC group (p<0.01). DHD differences were not significant in the HLU and WBC groups of the 14D and 90D/Recov time point. The 14D HLU DHD was a significantly higher than that of the 90D HLU and 90D/Recov groups (p<0.01 for both). Importantly, there were no significant differences in DHD between the 90D HLU and the 90D/Recov HLU groups.

Discussion: BMD analysis revealed a significant decrease in the 90D HLU group compared to the 14D HLU group and a significant recovery in BMD in the 90D/Recov group compared to the 90D HLU group. The initial decrease in BMD agreed with previous studies that revealed a similar decrease in mice sent on a 15-day space mission [12]. BMD and bone morphological parameters generally showed a significant difference between the 90D HLU and the 90D/Recov HLU groups. Furthermore, the lack of significant differences between the HLU and WBC groups of the 90D/Recov time point in BMD, BV/TV, Tr. Th, and Tr. Sp suggest that a recovery period can aid in bone recovery to baseline conditions. To examine the extent of bone recovery after hindlimb unloading, future analyses of the bone recovery at different time points will be needed. For IVD changes, significant DHD decreases were observed, especially in the lower lumbar regions of the 14D and 90D HLU groups. This agreed with previous data that reported a significant decrease in DHD in four-week himdlimb unloaded rats [13]. The lack of significant changes in DHD between the 90D HLU group and the 90D/Recov group indicated that removal of the microgravity environment may not be enough to aid in IVD recovery.

Significance: Extended unloading of the hindlimb results in decreased BMD, bone morphological parameters, and progressive decrease in IVD height. The 28-day recovery period, where the hindlimb unloading was removed, aided in bone recovery but was ineffective in IVD height restoration.

References:

(1) Johnston+. Aviat Space Environ Med, 81(6):566, 2010. (2) Orwoll+. JBMR, 28(6):1243, 2013. (3) Lang+. JBMR, 19(6):1006, 2004. (4) LeBlanc+. Bone, 22(5 Suppl):113S, 1998. (5) Turner+. J Appl Physiol, 89(2):840, 2000. (6) Morey-Holton+. J Appl Physiol, 92, 1367, 2002. (7) Canciani+. JMBBM, 51:1, 2015. (8) Cheng+, ORS Trans; 0696, 2015. (9) Tavella+. PLoS ONE, 7(3): e33179, 2012. (10) Buie+. Bone, 41(4):505, 2007. (11) Wantanabe+. Eur Spine J, 21(5):946, 2012. (12) Blaber+. PLoS One, 8(4):e61372, 2013. (13) Holguin, Aviat Space Environ Med, 81(12):1078

Other tests such as biomechanics and histomorphology will be delayed due to the unexpected slow arrival of samples.

Bibliography Type: Description: (Last Updated: 12/08/2021)  Show Cumulative Bibliography Listing
 
Abstracts for Journals and Proceedings Huang J, Cheng K, Kato K, Sah RL, Akeda K, Macias B, Inoue N, Hargens A, Masuda K. "28-Day Recovery After Microgravity Conditions Restored Rodent Bone Quality But Not Intervertebral Disc Height." Presented at 63rd Annual Meeting of the Orthopaedic Research Society, San Diego, CA, March 19-22, 2017.

63rd Annual Meeting of the Orthopaedic Research Society, San Diego, CA, March 19-22, 2017. , Mar-2017

Articles in Peer-reviewed Journals Hargens AR, Vico L. "Long-duration bed rest as an analog to microgravity." J Appl Physiol (1985). 2016 Apr 15;120(8):891-903. Review. http://dx.doi.org/10.1152/japplphysiol.00935.2015 ; PubMed PMID: 26893033 , Apr-2016
Project Title:  Spinal Structure and Function after 90 Days Long-Duration Simulated Space Flight and Recovery Reduce
Fiscal Year: FY 2016 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 08/01/2014  
End Date: 07/31/2017  
Task Last Updated: 06/08/2016 
Download report in PDF pdf
Principal Investigator/Affiliation:   Hargens, Alan R. Ph.D. / University of California, San Diego 
Address:  Altman Clinical and Translational Research Institute 
9452 Medical Center Drive/0863 
La Jolla , CA 92037-0863 
Email: ahargens@ucsd.edu 
Phone: 858-534-7837  
Congressional District: 52 
Web:  
Organization Type: UNIVERSITY 
Organization Name: University of California, San Diego 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Ferguson, Adam  Ph.D. University of California at San Francisco 
Lotz, Jeffrey  Ph.D. University of California at San Francisco 
Macias, Brandon  NASA Johnson Space Center 
Masuda, Koichi  M.D. University of California at San Diego 
Project Information: Grant/Contract No. NNX14AP25G 
Responsible Center: NASA JSC 
Grant Monitor: Norsk, Peter  
Center Contact:  
Peter.norsk@nasa.gov 
Solicitation / Funding Source: 2013 HERO NNJ13ZSA002N-Crew Health (FLAGSHIP & NSBRI) 
Grant/Contract No.: NNX14AP25G 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:  
No. of Bachelor's Candidates:
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:  
Human Research Program Elements: (1) HHC:Human Health Countermeasures
Human Research Program Risks: (1) Osteo:Risk Of Early Onset Osteoporosis Due To Spaceflight (No longer used, July 2020)
Human Research Program Gaps: (1) Osteo04:We do not know the contribution of each risk factor on bone loss and recovery of bone strength, and which factors are the best targets for countermeasure application (IRP Rev E)
Task Description: The vertebral bodies and flexible intervertebral discs are important, weight-bearing tissues that have adapted to gravitational stress. Consequently, the absence of gravitational axial loads during exposure to microgravity likely disrupts normal spine physiology. Throughout longer space flight missions, deconditioning of the intervertebral discs and spinal muscles poses a serious injury risk upon re-exposure to upright posture in a gravitational environment. We will use state-of-the-art technologies to quantify morphology, biochemistry, and kinematics of spines (including the vertebrae, intervertebral discs, and spinal muscles) of rats at defined time points as described in the NASA research announcement. After successful completion of our investigation, we will deliver a comprehensive database of simulated microgravity-induced spinal adaptations (type and magnitude). The overarching goal of these proposed studies are to develop a long-duration space flight ground based model of spine function and structure. In addition, this research project will afford the opportunity to examine possible gender differences in spinal structure and function. Our research group is in a unique position to leverage our past rodent space flight experience on STS-131, STS-133, STS-135, and BION M-1 missions and directly compare to this ground based model of simulated microgravity. Moreover, we are also uniquely positioned to compare this 90-days hindlimb suspension model to those changes that occur in our currently funded project to test crew members before and after 6-month International Space Station (ISS) missions. Our project directly addresses Critical Path Roadmap Risks and Questions regarding disc injury (IRP Gap-B4): Is damage to joint structure, intervertebral discs, or ligaments incurred during or following microgravity exposure? Our research will improve understanding of the underlying pathophysiology of spinal deconditioning induced by simulated microgravity, and mechanisms of spinal adaptation following re-exposure to 1-G. Our long-term goal is to prevent such spinal deconditioning with exercise or other physiologic countermeasures. The goal of this research is to comprehensively characterize 90-days simulated space flight and recovery induced changes in spinal tissue morphology, biochemistry, and biomechanics.

Research Impact/Earth Benefits: To our knowledge, this study is the first to examine the effects of 90-days simulated space flight on spinal deconditioning in rats and to compare this model of simulated microgravity with actual space flight. The vertebral bodies and flexible intervertebral discs are important, weight-bearing tissues that have adapted to gravitational stress. Our research will aid understanding of spinal deconditioning during simulated microgravity and of the higher incidence of disc prolapse or herniation following re-exposure to 1-G with a long-term view to prevent such spinal deconditioning with exercise or other physiologic countermeasures. This research may aid understanding of spinal deconditioning during inactivity such as after spinal cord injury and bed rest in human patients on Earth.

Task Progress & Bibliography Information FY2016 
Task Progress: To date we have received 39 hind-limb suspension (HLS) spine samples. Currently, all 24 of the 90-day HLS and 90-day HLS + 28-day recovery samples have already been µCT scanned at 9 µm resolution. There are a total of 13 spines remaining to scan, which include samples in the 14-day and 28-day HLS groups.

An abstract titled “90-Days Simulated Spaceflight Impairs Quality of Rodent Lumbar Intervertebral Disc and Bone” was presented at the Orthopaedic Research Society 2016 Annual Meeting in Orlando, FL. Data on the bone mineral density (BMD) and intervertebral disc (IVD) height changes in the 90-day HLS group was reported. BMD analysis reported significantly lower BMD in L1, L2, and L3 vertebral bodies compared to the weight-bearing controls (WC) (-20.6%, p<0.0001). BMD in L4-L5 in the HLS group was 10.3% lower; however, these decreases were not statistically significant. Overall, there was a 15.5% decrease in BMD in the HLS group compared to the WC group (p<0.0001). IVD height analysis found significantly decreased IVD height at L5/6 and L6/S1 by 6.4% (p=0.0467). However, IVD heights from Th13/L1 to L4/5 tended to be lower by 4.9%; but this was not statistically significant (p=0.3031). Across all levels, there was a 3.1% decrease in IVD height.

Currently, bone morphological analyses are underway. The analyses will yield measures such as percent bone volume, trabecular separation, trabecular thickness, bone surface volume ratio, bone surface density, etc. Because of the large dataset due to the high resolution scans, an optimized analysis method utilizing Ct-Analyser (Bruker, Kontich, Belgium) and Matlab (Mathworks, Natick, MA) is currently being made.

Other tests such as biomechanics and histomorphology will be delayed due to the unexpected slow arrival of samples.

Bibliography Type: Description: (Last Updated: 12/08/2021)  Show Cumulative Bibliography Listing
 
Abstracts for Journals and Proceedings Cheng K, Macias BR, Hargens A, Masuda K. "90-Days Simulated Spaceflight Impairs Quality of Rodent Lumbar Intervertebral Disc and Bone." Presented at 62nd Annual Meeting of the Orthopaedic Research Society, Orlando, FL, March 5-8, 2016.

62nd Annual Meeting of the Orthopaedic Research Society, Orlando, FL, March 5-8, 2016. Orthopaedic Research Society abstracts. Poster No. 0696. , Mar-2016

Articles in Peer-reviewed Journals Berg-Johansen B, Liebenberg EC, Li A, Macias BR, Hargens AR, Lotz JC. "Spaceflight-induced bone loss alters failure mode and reduces bending strength in murine spinal segments." J Orthop Res. 2016 Jan;34(1):48-57. Epub 2015 Aug 31. http://dx.doi.org/10.1002/jor.23029 ; PubMed PMID: 26285046 , Jan-2016
Project Title:  Spinal Structure and Function after 90 Days Long-Duration Simulated Space Flight and Recovery Reduce
Fiscal Year: FY 2015 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 08/01/2014  
End Date: 07/31/2017  
Task Last Updated: 04/26/2016 
Download report in PDF pdf
Principal Investigator/Affiliation:   Hargens, Alan R. Ph.D. / University of California, San Diego 
Address:  Altman Clinical and Translational Research Institute 
9452 Medical Center Drive/0863 
La Jolla , CA 92037-0863 
Email: ahargens@ucsd.edu 
Phone: 858-534-7837  
Congressional District: 52 
Web:  
Organization Type: UNIVERSITY 
Organization Name: University of California, San Diego 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Ferguson, Adam  Ph.D. University of California at San Francisco 
Lotz, Jeffrey  Ph.D. University of California at San Francisco 
Macias, Brandon  NASA-JSC 
Masuda, Koichi  M.D. University of California at San Diego 
Project Information: Grant/Contract No. NNX14AP25G 
Responsible Center: NASA JSC 
Grant Monitor: Norsk, Peter  
Center Contact:  
Peter.norsk@nasa.gov 
Solicitation / Funding Source: 2013 HERO NNJ13ZSA002N-Crew Health (FLAGSHIP & NSBRI) 
Grant/Contract No.: NNX14AP25G 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:
No. of PhD Candidates:  
No. of Master's Candidates:  
No. of Bachelor's Candidates:
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:  
Human Research Program Elements: (1) HHC:Human Health Countermeasures
Human Research Program Risks: (1) Osteo:Risk Of Early Onset Osteoporosis Due To Spaceflight (No longer used, July 2020)
Human Research Program Gaps: (1) Osteo04:We do not know the contribution of each risk factor on bone loss and recovery of bone strength, and which factors are the best targets for countermeasure application (IRP Rev E)
Task Description: The vertebral bodies and flexible intervertebral discs are important, weight-bearing tissues that have adapted to gravitational stress. Consequently, the absence of gravitational axial loads during exposure to microgravity likely disrupts normal spine physiology. Throughout longer space flight missions, deconditioning of the intervertebral discs and spinal muscles poses a serious injury risk upon re-exposure to upright posture in a gravitational environment. We will use state-of-the-art technologies to quantify morphology, biochemistry, and kinematics of spines (including the vertebrae, intervetebral discs, and spinal muscles) of rats at defined time points as described in the NASA research announcement. After successful completion of our investigation, we will deliver a comprehensive database of simulated microgravity-induced spinal adaptations (type and magnitude). The overarching goal of these proposed studies are to develop a long-duration space flight ground based model of spine function and structure. In addition, this research project will afford the opportunity to examine possible gender differences in spinal structure and function. Our research group is in a unique position to leverage our past rodent space flight experience on STS-131, STS-133, STS-135, and BION M-1 missions and directly compare to this ground based model of simulated microgravity. Moreover, we are also uniquely positioned to compare this 90-days hind-limb suspension model to those changes that occur in our currently-funded project to test crew members before and after 6-month ISS missions. Our project directly addresses Critical Path Roadmap Risks and Questions regarding disc injury (IRP Gap-B4): Is damage to joint structure, intervertebral discs, or ligaments incurred during or following microgravity exposure? Our research will improve understanding of the underlying pathophysiology of spinal deconditioning induced by simulated microgravity, and mechanisms of spinal adaptation following re-exposure to 1-G. Our long-term goal is to prevent such spinal deconditioning with exercise or other physiologic countermeasures. The goal of this research is to comprehensively characterize 90-days simulated space-flight and recovery induced changes in spinal tissue morphology, biochemistry, and biomechanics.

Research Impact/Earth Benefits: To our knowledge, this study is the first to examine the effects of 90-days simulated space flight on spinal deconditioning in rats. The vertebral bodies and flexible intervertebral discs are important, weight-bearing tissues that have adapted to gravitational stress. Our research will aid understanding of spinal deconditioning during simulatee microgravity and of the higher incidence of disc prolapse or herniation following re-exposure to 1-G with a long-term view to prevent such spinal deconditioning with exercise or other physiologic countermeasures. This research may aid understanding of spinal deconditioning during inactivity such as after spinal cord injury and bed rest in human patients on Earth.

Task Progress & Bibliography Information FY2015 
Task Progress: Limited progress has been made because we are just now receiving spine samples from the Body Parts Program at UC-Davis/NASA Ames Research Center.

Bibliography Type: Description: (Last Updated: 12/08/2021)  Show Cumulative Bibliography Listing
 
 None in FY 2015
Project Title:  Spinal Structure and Function after 90 Days Long-Duration Simulated Space Flight and Recovery Reduce
Fiscal Year: FY 2014 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 08/01/2014  
End Date: 07/31/2017  
Task Last Updated: 09/18/2014 
Download report in PDF pdf
Principal Investigator/Affiliation:   Hargens, Alan R. Ph.D. / University of California, San Diego 
Address:  Altman Clinical and Translational Research Institute 
9452 Medical Center Drive/0863 
La Jolla , CA 92037-0863 
Email: ahargens@ucsd.edu 
Phone: 858-534-7837  
Congressional District: 52 
Web:  
Organization Type: UNIVERSITY 
Organization Name: University of California, San Diego 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Ferguson, Adam  Ph.D. University of California at San Francisco 
Lotz, Jeffrey  Ph.D. University of California at San Francisco 
Macias, Brandon  Ph.D. University of California at San Diego 
Masuda, Koichi  M.D. University of California at San Diego 
Project Information: Grant/Contract No. NNX14AP25G 
Responsible Center: NASA JSC 
Grant Monitor: Norsk, Peter  
Center Contact:  
Peter.norsk@nasa.gov 
Solicitation / Funding Source: 2013 HERO NNJ13ZSA002N-Crew Health (FLAGSHIP & NSBRI) 
Grant/Contract No.: NNX14AP25G 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:  
No. of PhD Candidates:  
No. of Master's Candidates:  
No. of Bachelor's Candidates:  
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:  
Human Research Program Elements: (1) HHC:Human Health Countermeasures
Human Research Program Risks: (1) Osteo:Risk Of Early Onset Osteoporosis Due To Spaceflight (No longer used, July 2020)
Human Research Program Gaps: (1) Osteo04:We do not know the contribution of each risk factor on bone loss and recovery of bone strength, and which factors are the best targets for countermeasure application (IRP Rev E)
Task Description: The vertebral bodies and flexible intervertebral discs are important, weight-bearing tissues that have adapted to gravitational stress. Consequently, the absence of gravitational axial loads during exposure to microgravity likely disrupts normal spine physiology. Throughout longer space flight missions, deconditioning of the intervertebral discs and spinal muscles poses a serious injury risk upon re-exposure to upright posture in a gravitational environment. We will use state-of-the-art technologies to quantify morphology, biochemistry, and kinematics of spines (including the vertebrae, intervetebral discs, and spinal muscles) of rats at defined time points as described in the NASA research announcement. After successful completion of our investigation, we will deliver a comprehensive database of simulated microgravity-induced spinal adaptations (type and magnitude). The overarching goal of these proposed studies are to develop a long-duration space flight ground based model of spine function and structure. In addition, this research project will afford the opportunity to examine possible gender differences in spinal structure and function. Our research group is in a unique position to leverage our past rodent space flight experience on STS-131, STS-133, STS-135, and BION M-1 missions and directly compare to this ground based model of simulated microgravity. Moreover, we are also uniquely positioned to compare this 90-days hind-limb suspension model to those changes that occur in our currently-funded project to test crew members before and after 6-month ISS missions. Our project directly addresses Critical Path Roadmap Risks and Questions regarding disc injury (IRP Gap-B4): Is damage to joint structure, intervertebral discs, or ligaments incurred during or following microgravity exposure? Our research will improve understanding of the underlying pathophysiology of spinal deconditioning induced by simulated microgravity, and mechanisms of spinal adaptation following re-exposure to 1-G. Our long-term goal is to prevent such spinal deconditioning with exercise or other physiologic countermeasures. The goal of this research is to comprehensively characterize 90-days simulated space-flight and recovery induced changes in spinal tissue morphology, biochemistry, and biomechanics.

Research Impact/Earth Benefits: 0

Task Progress & Bibliography Information FY2014 
Task Progress: New project for FY2014.

Bibliography Type: Description: (Last Updated: 12/08/2021)  Show Cumulative Bibliography Listing
 
 None in FY 2014