NOTE: Extended to 8/31/2017 per NSSC information (Ed., 1/24/17)

NOTE: Extended to 10/05/2016 per NSSC information (Ed., 10/28/15)

Sclerostin is produced by osteocytes, the bone cells embedded in bone matrix and the key sensors of loading/unloading; it works to inhibit the Wnt-Beta catenin signaling pathway in osteoblasts, which normally stimulates bone formation activity. Most studies to date document an increase in osteocyte’s sclerostin expression with unloading, which provides the mechanism for the suppressed bone formation seen with HLS on Earth and, presumably, with microgravity exposure. A recent study examining the impact of a novel therapeutic agent (sclerostin antibody) on mice flown on STS-135 yielded very positive findings, suggesting that manipulating sclerostin expression could be an important therapeutic tool to augment the usual exercise countermeasures employed by astronauts. Hence it becomes critical, before any such systemic therapy is considered, to understand clearly the relationship between sclerostin expression and the functionally important outcome (bone formation activity) in multiple bone sites.

Because there is evidence that Sost, the gene encoding the protein sclerostin, is expressed differently in mid-shaft vs metaphyseal bone, we will assess sclerostin expression and histomorphometric measures of bone formation in 3 different bone compartments for each bone: mid-shaft cortical bone, cancellous bone of the metaphysis and the metaphyseal cortical shell. We propose to first study these outcomes in paired tibiae or femurs (unloaded bone) and in normally loaded humeri. With the availability of female mice in the parent study’s 2nd specific aim, we can then assess if there are gender differences in this response. Recently published data on spaceflown mice document an increase in bone volume in calvarial bone, raising the intriguing possibility that fluid shifts during spaceflight may increase fluid pressures in the rodent brain compartment, providing a mechanical loading of sorts to the skull. Hence, a third specific aim of our proposal, pending verification that calvarial bone can be made available, will assess sclerostin expression and bone formation variables in this unique site to test this hypothesis. At the end of one year’s intensive effort, then, we will have gained a much more clear picture of how this important regulatory molecule is altered by unloading and whether sclerostin antibody does present a viable therapeutic tool for maintaining bone integrity on long-duration missions. In addition, we will gain important fundamental knowledge about the time course of sclerostin expression and its relationship to bone formation rate with alterations in mechanical loading.

The only timepoint for which we received adequate numbers of samples from the female cohorts was at 14 d HU; hence, we were able to complete statistical analyses of sex differences for that one time point. These were the planned analyses; some are still in progress :

A. Peripheral quantitative computed tomography (for BMD, bone geometry)

a. Young males at 7, 14, 28, and 90 d of HU and 14, 28, and 90 d of recovery from HU

b. Old males at 14 and 90 d HU and 14 and 90 d of recovery from HU

c. Young females at 14 d HU

B. Static histomorphometry for % osteoid (new bone; index of formation activity) and for % osteoclast (bone resorbing) surfaces; cancellous bone volume and microarchitecture

a. Young males at 7, 14, 28, and 90 d of HU and 14, 28, and 90 d of recovery from HU

b. Old males at 14 d and 90 d of HU and 14 and 90 d of recovery from HU (still in progress)

c. Young females at 14 d HU

C. Immunohistochemistry staining for osteocyte sclerostin

a. Young males at 14 and 90 d of HU and 14 and 90 d of weightbearing (WB) recovery from HU

b. Old males at 14 d and 90 d of HU and 14 and 90 d of weightbearing recovery from HU (still in progress)

c. Young females at 14 d HU

Summary of key findings (organized under original hypotheses): H1: Increases in Scl-IHC in osteocytes of cortical mid-shaft bone will be observed by 4 days of HLS in mid-shaft cortical bone, preceding a decline in formation indices. These increases will remain high throughout the period of HLS.

H2: Scl-IHC in metaphyseal bone osteocytes (cortical shell and cancellous core) will follow a similar pattern as in mid-shaft osteocytes, but increases will be delayed till 7 days of HLS or later.

Information in NASA Human Research Program (HRP) meeting 2016 poster: At 14 d of HU there was no detectable elevation of % sclerostin-positive osteocytes in any of 3 bone compartments analyzed (mid-shaft cortical bone, cortical shell and cancellous core of femur metaphysis) in young male rats. An unusual 3-fold elevation of % osteoid surface (our one bone formation index) was observed at this time point. We did observe a small decline over 14 d HU in total/integral and cancellous volumetric bone mineral density (vBMD) at the proximal tibia metaphysis (by pQCT measures). [NASA HRP 2016 poster:] We did observe ~50% increase in %sclerostin-positive osteocytes at 90 d HU but only in cancellous bone compartment. At this same time point, % osteoid surface remained elevated above weightbearing controls. The same % deficit in total/integral and cancellous volumetric bone mineral density (vBMD) at the proximal tibia metaphysis that was observed after 14 d HU was observed at 90 d HU, suggesting a plateauing of bone loss in the latter stages of this prolonged unloading period.

H3: During the initial weightbearing recovery period, Scl-IHC will decline relative to peak HLS values in all bone compartments, preceding a local increase in formation indices. Scl-IHC and indices of bone formation will approach aging control values after 90 days of recovery.

Answering this hypothesis awaits completion of sclerostin protein immunostaining osteocytes in recovery samples.

H4: Relative changes in Scl-IHC and indices of bone formation, and the timing thereof, during HLS and recovery in young female rats will not be different from those observed in young male rats.

Information in NASA HRP 2017 poster: There were no differences in the prevalence of %sclerostin-positive osteocytes between male and female rats after 14 d HU. However, females did not exhibit the increase in % osteoid surface as observed in males at this time point (no change vs. WB controls); they also exhibited a 25% increase in %osteoclast surface. Interestingly, pQCT scans could not detect declines in total/integral and cancellous vBMD at the proximal tibia metaphysis in these female rats as was observed in males. Histological (and likely more precise evaluation) of cancellous bone volume revealed a significant decline in females but not males.

H5: Relative changes in Scl-IHC and indices of bone formation will be smaller in these older male rats (9-mo-old) with unloading and a return to weightbearing, but the relationship between magnitude of change in Scl-IHC and bone formation will not be altered by aging.

Answering this hypothesis awaits completion of sclerostin immunostaining in older male rat samples.

Information in Texas Chapter of American College of Sports Medicine (ACSM) 2017 poster: Older (9-month-old) male rats do not experience the significant declines (12-26%) in total/integral and cancellous vBMD at the proximal tibia, nor the decline in cancellous bone volume and trabecular number (by histomorphometry) at the proximal tibia metaphysis that were observed in young (3-month-old) rats after 14 days of hindlimb unloading. This could result, in part, from the fact that older male weightbearing controls have much lower values for these parameters than do the young controls, suggesting there is a lower inherent limit to these values.

NOTE: Extended to 10/05/2016 per NSSC information (Ed., 10/28/15)

Sclerostin is produced by osteocytes, the bone cells embedded in bone matrix and the key sensors of loading/unloading; it works to inhibit the Wnt-Beta catenin signaling pathway in osteoblasts, which normally stimulates bone formation activity. Most studies to date document an increase in osteocyte’s sclerostin expression with unloading, which provides the mechanism for the suppressed bone formation seen with HLS on Earth and, presumably, with microgravity exposure. A recent study examining the impact of a novel therapeutic agent (sclerostin antibody) on mice flown on STS-135 yielded very positive findings, suggesting that manipulating sclerostin expression could be an important therapeutic tool to augment the usual exercise countermeasures employed by astronauts. Hence it becomes critical, before any such systemic therapy is considered, to understand clearly the relationship between sclerostin expression and the functionally important outcome (bone formation activity) in multiple bone sites.

Because there is evidence that Sost, the gene encoding the protein sclerostin, is expressed differently in mid-shaft vs metaphyseal bone, we will assess sclerostin expression and histomorphometric measures of bone formation in 3 different bone compartments for each bone: mid-shaft cortical bone, cancellous bone of the metaphysis and the metaphyseal cortical shell. We propose to first study these outcomes in paired tibiae or femurs (unloaded bone) and in normally loaded humeri. With the availability of female mice in the parent study’s 2nd specific aim, we can then assess if there are gender differences in this response. Recently published data on spaceflown mice document an increase in bone volume in calvarial bone, raising the intriguing possibility that fluid shifts during spaceflight may increase fluid pressures in the rodent brain compartment, providing a mechanical loading of sorts to the skull. Hence, a third specific aim of our proposal, pending verification that calvarial bone can be made available, will assess sclerostin expression and bone formation variables in this unique site to test this hypothesis. At the end of one year’s intensive effort, then, we will have gained a much more clear picture of how this important regulatory molecule is altered by unloading and whether sclerostin antibody does present a viable therapeutic tool for maintaining bone integrity on long-duration missions. In addition, we will gain important fundamental knowledge about the time course of sclerostin expression and its relationship to bone formation rate with alterations in mechanical loading.

We received our first shipment of bones from the parent protocol at UC-Davis in early April of 2015 (n ~ 43) from young adult male rats, including samples from animals sacrificed after 7, 14, 28, and 90 days of hindlimb unloading, as well as after 28 days of weight-bearing recovery. Our revised protocol submitted in August, 2014, after negotiations with Dr. Peter Norsk and the parent protocol Principal Investigator (PI) (C Fuller), described proposed work we would perform on the tibial bone samples targeted to our laboratory for analysis. Our first task was to run ex vivo pQCT scans to quantitate bone structural changes on both the metaphysis, a site very sensitive to disuse, and the mid-shaft bone. Outcomes here include volumetric bone mineral density (vBMD), bone mineral content, and cross-sectional geometry (e.g., bone area, cortical thickness, marrow area). Once these were complete, the proximal half of one tibia was prepared histologically for embedding in hard plastic in order for us to perform standard static histomorphometry on the proximal tibial metaphysis. Quantitating the extent of surfaces covered by newly formed bone matrix (osteoid) and by osteoclasts (bone resorbing cells) provides useful information on the balance between bone forming and bone resorbing activity. [Because the parent protocol PI was reticent to add more procedures to an already complicated protocol, there was no injection of fluorochrome labels in these rats during the experiments at UC-Davis, which disallows determination of bone formation rate.]

Summary of results: To date, 2-month old male rats exposed to hindlimb unloading (HLU) demonstrate the expected reductions in vBMD at the proximal tibia metaphysis, as compared to age-matched controls at both 14 and 90 days of HLU. The trend of bone loss appears to remain consistent from 7 days through to recovery (28 days of weight bearing recovery following 90 days of HU). Our histomorphometry results thus far are not consistent with these structural changes in bone described by the pQCT data. Despite the bone loss, there is a paradoxical increase in osteoid surface (newly formed bone matrix yet to be mineralized) at both 14 days and 90 days with the suggestion of similar trends at both 28 days HLU and 28 days of recovery. Likewise, there is a decrease in osteoclast surface (a measure of bone resorption) in HLU at 14 days with no different between HLU and CC at 90 days. Limitations of our current work with this study include the low animal numbers at all time points except 14 days and 90 days, making it difficult to draw firm conclusions of the time course of HLU changes. These preliminary data will be presented at the NASA Human Research Program (HRP) Investigator Workshop in February 2016.

Sclerostin is produced by osteocytes, the bone cells embedded in bone matrix and the key sensors of loading/unloading; it works to inhibit the Wnt- Beta catenin signaling pathway in osteoblasts, which normally stimulates bone formation activity. Most studies to date document an increase in osteocyte’s sclerostin expression with unloading, which provides the mechanism for the suppressed bone formation seen with HLS on Earth and, presumably, with microgravity exposure. A recent study examining the impact of a novel therapeutic agent (sclerostin antibody) on mice flown on STS-135 yielded very positive findings, suggesting that manipulating sclerostin expression could be an important therapeutic tool to augment the usual exercise countermeasures employed by astronauts. Hence it becomes critical, before any such systemic therapy is considered, to understand clearly the relationship between sclerostin expression and the functionally important outcome (bone formation activity) in multiple bone sites.

Because there is evidence that Sost, the gene encoding the protein sclerostin, is expressed differently in mid-shaft vs metaphyseal bone, we will assess sclerostin expression and histomorphometric measures of bone formation in 3 different bone compartments for each bone: mid-shaft cortical bone, cancellous bone of the metaphysis and the metaphyseal cortical shell. We propose to first study these outcomes in paired tibiae or femurs (unloaded bone) and in normally loaded humeri. With the availability of female mice in the parent study’s 2nd specific aim, we can then assess if there are gender differences in this response. Recently published data on spaceflown mice document an increase in bone volume in calvarial bone, raising the intriguing possibility that fluid shifts during spaceflight may increase fluid pressures in the rodent brain compartment, providing a mechanical loading of sorts to the skull. Hence, a third specific aim of our proposal, pending verification that calvarial bone can be made available, will assess sclerostin expression and bone formation variables in this unique site to test this hypothesis. At the end of one year’s intensive effort, then, we will have gained a much more clear picture of how this important regulatory molecule is altered by unloading and whether sclerostin antibody does present a viable therapeutic tool for maintaining bone integrity on long-duration missions. In addition, we will gain important fundamental knowledge about the time course of sclerostin expression and its relationship to bone formation rate with alterations in mechanical loading.