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Project Title:  Contribution of the Vestibular and Sympathetic Nervous Systems to Space-Induced Bone Loss (Postdoctoral Fellowship) Reduce
Fiscal Year: FY 2014 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 11/01/2011  
End Date: 10/31/2013  
Task Last Updated: 02/06/2014 
Download report in PDF pdf
Principal Investigator/Affiliation:   Vignaux, Guillaume F. Ph.D. / Vanderbilt University 
Address:  Center for Bone Biology 
1211 Medical Center Drive 
Nashville , TN 37232 
Email: guillaume.f.vignaux@vanderbilt.edu 
Phone: 615-322-7883  
Congressional District:
Web:  
Organization Type: UNIVERSITY 
Organization Name: Vanderbilt University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Elefteriou, Florent  MENTOR/ Vanderbilt University 
Project Information: Grant/Contract No. NCC 9-58-PF02603 
Responsible Center: NSBRI 
Grant Monitor:  
Center Contact:   
Solicitation / Funding Source: 2011 NSBRI-RFA-11-01 Postdoctoral Fellowships 
Grant/Contract No.: NCC 9-58-PF02603 
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: POSTDOCTORAL FELLOWSHIP

Our main hypothesis is that the vestibular system participates to the maintenance of bone mineral density on Earth and its dysfunction under microgravity may contribute to the bone loss associated with space travel. Our preliminary findings have provided evidence to support our hypothesis. Bilateral vestibular lesion (VBX) using sodium arsanilate injections in rats led to significant bone loss associated to a decrease in osteoblasts number.

Aims of this project are 1) to determine if VBX causes bone loss by activation of the sympathetic nervous system in our VBX model using beta-blocker-treated mice and mice lacking the beta-2 adrenergic receptors globally or specifically in osteoblasts; 2) to analyze the bone phenotype of mice devoid of vestibular gravity sensor (Het-/- mice), and 3) to test nitric oxide involvement in vestibular-related bone loss using a vestibular hair cells specific KO for Sod3. Our study may uncover a new pathway of bone regulation, a novel approach for the treatment of low bone mass diseases on Earth, and novel countermeasures to reduce risk of bone fracture in microgravity.

During our first year of research we confirmed our previous results obtained in rats in a mouse model of vestibular lesion. Indeed 2-month old mice displayed reduced bone mineral density in femurs 1 month after VBX, as observed in rats. This result is important as it allows us to use genetically-modified mutant mice in future studies. We also investigated the SNS involvement in VBX-induced bone loss in mice. We found that daily propranolol treatment prevented VBX-induced bone loss, as it did previously in rats, and beta-2 adrenergic receptors KO mice femurs were resistant to VBX. Both results support the hypothesis of a SNS involvement in our model. We went further by using mice lacking the beta-2 adrenergic receptors specifically on osteoblasts. These mice were also resistant to VBX showing that the VBX-induced bone loss is a SNS mediated effect acting through the osteoblasts. Finally, no bone change was observed in 3-month old Het–/– mice (Nox3het/J) (mice lacking otoliths). Nox3 encodes a NADPH oxidase which is involved in the down-regulation of nitric oxide (NO) availability. Knowing that NO level in vestibular hair cells modulates vestibular signals and that it is an important neuromediator/neuromodulator of the vestibular response, we hypothesized that the combination of lack of otoliths and impaired neurotransmission possibly impact bone remodeling in a manner that is more complicated than anticipated.

Aim 3 of this project was aimed at teasing apart the mechanism involved. We are currently breeding Sod3 flox mice that, in addition to results of aim 2, will help us to better understand the role of NO in vestibular response generation in hair cells.

Research Impact/Earth Benefits: Until 2012 there was no published data regarding the role of the vestibular system in bone biology. However, the presence of sympathetic nerves in the bone environment was reported more than 50 years ago (PMID: 13525457 ) and our laboratory has more recently shown, using genetic and pharmacologic approaches, that sympathetic nerves contribute to the regulation of bone homeostasis (PMID: 15724149 ). Osteoblasts express the beta-2 adrenergic receptor (ß2AR) and respond to catecholamines or pharmacological ßAR agonists with decreased proliferation (PMID: 18410742 ) and induction of RANKL expression, which leads to enhanced osteoclast formation and bone resorption. Propranolol, a ß1/ß2AR non-selective antagonist, inhibits these effects of sympathetic activation and protects from ovariectomy and unloading-induced bone loss in mice and rats (PMID: 12419242 , PMID: 15961387 , PMID: 17243867 ); in several retrospective studies, the use of ß-blockade was associated with increased BMD and decreased fracture rate in humans (PMID: 15724149 , PMID: 1919850 , PMID: 18622078 ). External factors also influence bone remodeling. In particular, gravity and inertial accelerations exert a range of mechanical stimulations on the skeleton that have an osteogenic effect demonstrated in birds, mice, rats, pigs, sheep and humans (PMID: 11541937 , PMID: 7153230 , PMID: 6699056 , PMID: 8368304 , PMID: 17185839 ). In contrast, mechanical unloading as a consequence of bed-rest or microgravity conditions causes bone loss in humans (PMID: 17396004 ) and rodents (PMID: 11541702 ), which is associated with suppressed bone formation and a mild increase in resorption. Weight-bearing pressure is sensed by proprioceptive sensors in the joints, capsules, ligaments, muscles, tendons and skin, while the gravito-inertial acceleration (generating the weight) is sensed by otoliths and visceral graviceptors (PMID: 22579804 ). The otolith system, which is part of the vestibular system in the inner ear, is the main sensory organ of gravity and linear accelerations. Its contribution to the regulation of posture, respiration, heart rate and blood pressure in animals (PMID: 12787870 , PMID: 14660474 , PMID: 15640755 , PMID: 21921247 ) and humans (PMID: 9173934 , PMID: 12111293 ) is well-documented and supported by anatomical projections from vestibular nuclei to autonomic centers of the brainstem (PMID: 7611512 , PMID: 18809392 ). Vestibular inputs contribute to changes in sympathetic nerve outflow during movements and postural changes that follow stimulation of the vestibulo-sympathetic reflex (PMID: 9728081 , PMID: 9416585 , PMID: 10896872 , PMID: 22946097 ), as demonstrated by the orthostatic hypotension observed in cats with bilateral vestibular destruction (PMID: 15475594 ). In 2013 our laboratory reported evidence for a role of the vestibular system in bone homeostasis control in rats (PMID: 23553797 ). Using an established model of bilateral vestibular lesions and microtomographic and histomorphometric bone analyses, we showed that induction of bilateral vestibular lesion in rats generates significant bone loss, which is restricted to weight-bearing bones and associated with a significant reduction in bone formation. Importantly, this bone loss was not associated with reduced locomotor activity or metabolic abnormalities, was accompanied with molecular signs of increased sympathetic outflow, and could be prevented by the ß-blocker propranolol. Collectively, these data suggest that the homeostatic process of bone remodeling has a vestibulo-sympathetic regulatory component and that vestibular system pathologies might be accompanied by bone fragility. The vestibular system can be affected in several ways. Thus our work might be helpful for patients after direct damages of the vestibular system, such as idiopathic vestibular areflexia, vestibular neuritis, Menière's disease, labyrinthectomy, vestibular nerve section, or the use of ototoxic antibiotics, or with a vestibular disorder subsequent to an ischemic stroke, a neurotoxic pathology or a neural tumor.

Task Progress & Bibliography Information FY2014 
Task Progress: SPECIFIC AIM 1: Determine if vestibular lesion causes bone loss by activation of the sympathetic nervous system. - The first step of this study consisted in evaluating the effect of a bilateral vestibular lesion (VBX) on femoral bone mass. Using micro-CT analyses on 2-month old mice, we demonstrated that VBX induces a significant decrease in femoral bone mass one month after the lesions. Histomorphometric analyses revealed that this bone loss was associated with a decrease in bone formation whereas bone resorption was not affected. - The second step was to determine whether the sympathetic nervous system (SNS) mediates this VBX-induced bone loss. Using 2-month old beta-2 adrenergic receptors KO mice, we detected no bone change in BV/TV 1 month after VBX. Moreover, daily propranolol treatment had no effect on WT-sham mice but completely blunted VBX-induced bone loss in WT-VBX mice. Finally the SNS involvement in the VBX-induced bone loss was further supported by the increase in Ucp1 gene expression in brown adipose tissue (a marker of the SNS activity). - No change in BV/TV was observed 1 month after VBX in 2-month old beta-2 adrenergic receptors osteoblasts-specific KO mice. These results suggest that VBX-induced sympathetic outflow to bone triggers bone loss via its effect on beta-2 adrenergic receptors expressing osteoblasts.

SPECIFIC AIM 2: Analyze the bone phenotype of mice devoid of vestibular gravity sensor. We used 3-month old Het–/– mice (Nox3het/J), lacking otoliths (gravity sensors), in order to mimic the decrease in vestibular stimulation in space. Micro-CT analyses revealed no bone changes in these mice compared to WT. Because of the lack of otoliths, we assumed that these mice should present a decrease in vestibular inputs and bone loss. However, Nox3 encodes a NADPH oxidase in hair cells which is involved in the downregulation of nitric oxide (NO) availability. Knowing that NO level in vestibular hair cells modulates the vestibular signals and that it is an important neuromediator/neuromodulator of the vestibular response, a constitutive high NO level in hair cells might explain our results in this model (aim 3 will help to clarify this). Another possible explanation is that the time point analyzed is not optimal. Younger and older mice will thus be analyzed.

SPECIFIC AIM 3: Test nitric oxide involvement in vestibular-related bone loss. The Sod3flox/flox mice are currently breeding. A cre-adenovirus will be injected in the inner ear to inactivate Sod3 specifically in the vestibular system. The bone phenotype will then be analyzed by micro-CT and histomorphometry. Tomato reporter mice are currently tested to track cre virus infection and identify optimal titer.

Bibliography Type: Description: (Last Updated: 04/12/2016)  Show Cumulative Bibliography Listing
 
Articles in Peer-reviewed Journals Vignaux G, Besnard S, Ndong J, Philoxène B, Denise P, Elefteriou F. "Bone remodeling is regulated by inner ear vestibular signals." J Bone Miner Res. 2013 Oct;28(10):2136-44. http://dx.doi.org/10.1002/jbmr.1940 ; PubMed PMID: 23553797 , Oct-2013
Articles in Peer-reviewed Journals Vignaux G, Besnard S, Denise P, Elefteriou F. "The vestibular system: a newly identified regulator of bone homeostasis acting through the sympathetic nervous system." Current Osteoporosis Reports. 2015 Aug;13(4):198-205. [2015 May 28. Epub ahead of print] http://dx.doi.org/10.1007/s11914-015-0271-2 ; PubMed PMID: 26017583 , Aug-2015
Articles in Peer-reviewed Journals Vignaux G, Ndong JD, Perrien DS, Elefteriou F. "Inner ear vestibular signals regulate bone remodeling via the sympathetic nervous system." J Bone Miner Res. 2015 Jun;30(6):1103-11. [2014 Dec 10. Epub ahead of print] http://dx.doi.org/10.1002/jbmr.2426 ; PubMed PMID: 25491117 , Jun-2015
Significant Media Coverage MacMillan L. "Inner ear's role in bone remodeling. Online article describing PostDoc's research." Research news@Vanderbilt, May 15, 2013. http://news.vanderbilt.edu/2013/05/inner-ear-role-bone-remodeling/ , May-2013
Project Title:  Contribution of the Vestibular and Sympathetic Nervous Systems to Space-Induced Bone Loss (Postdoctoral Fellowship) Reduce
Fiscal Year: FY 2013 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 11/01/2011  
End Date: 10/31/2013  
Task Last Updated: 11/16/2012 
Download report in PDF pdf
Principal Investigator/Affiliation:   Vignaux, Guillaume F. Ph.D. / Vanderbilt University 
Address:  Center for Bone Biology 
1211 Medical Center Drive 
Nashville , TN 37232 
Email: guillaume.f.vignaux@vanderbilt.edu 
Phone: 615-322-7883  
Congressional District:
Web:  
Organization Type: UNIVERSITY 
Organization Name: Vanderbilt University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Elefteriou, Florent  MENTOR/ Vanderbilt University 
Project Information: Grant/Contract No. NCC 9-58-PF02603 
Responsible Center: NSBRI 
Grant Monitor:  
Center Contact:   
Solicitation / Funding Source: 2011 NSBRI-RFA-11-01 Postdoctoral Fellowships 
Grant/Contract No.: NCC 9-58-PF02603 
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: POSTDOCTORAL FELLOWSHIP

Our main hypothesis is that the vestibular system participates to the maintenance of bone mineral density on Earth and its dysfunction under microgravity may contribute to the bone loss associated with space travel. Our preliminary findings have provided evidence to support our hypothesis. Bilateral vestibular lesion (VBX) using sodium arsanilate injections in rats led to significant bone loss associated to a decrease in osteoblasts number.

Aims of this project are 1) to determine if VBX causes bone loss by activation of the sympathetic nervous system in our VBX model using beta-blocker-treated mice and mice lacking the beta-2 adrenergic receptors globally or specifically in osteoblasts; 2) to analyze the bone phenotype of mice devoid of vestibular gravity sensor (Het-/- mice); and 3) to test nitric oxide involvement in vestibular-related bone loss using a vestibular hair cells specific KO for Sod3. Our study may uncover a new pathway of bone regulation, a novel approach for the treatment of low bone mass diseases on Earth, and novel countermeasures to reduce risk of bone fracture in microgravity.

During this first year of research we confirmed our previous results obtained in rats in a mouse model of vestibular lesion. Indeed 2-month old mice displayed reduced bone mineral density in femurs 1 month after VBX, as observed in rats. This first result is important as it allows us to use genetically-modified mutant mice in future studies. We also investigated the SNS involvement in VBX-induced bone loss in mice. We found that daily propranolol treatment prevented VBX-induced bone loss, as it did previously in rats, and beta-2 adrenergic receptors KO mice femurs were not sensitive to VBX. Both results support the hypothesis of a SNS involvement in our model. Finally, no bone change was observed in 3-month old Het–/– mice (Nox3het/J) (mice lacking otoliths). Nox3 encodes a NADPH oxidase which is involved in the down-regulation of nitric oxide (NO) availability. Knowing that NO level in vestibular hair cells modulates vestibular signals and that it is an important neuromediator/neuromodulator of the vestibular response, we hypothesize that the combination of lack of otoliths and impaired neurotransmission possibly impact bone remodeling in a manner that is more complicated than anticipated.

Aim 3 of this project will help teasing apart the mechanism involved. We are currently breeding conditional beta-2 adrenergic receptors KO mice lacking this receptor specifically on osteoblasts in order to confirm, genetically and without the possible complications of developmental phenotypes, the SNS involvement in VBX-induced bone loss. We will also start aim 3 of our project which, in addition to results of aim 2, will help us to better understand the role of NO in vestibular response generation in hair cells.

Research Impact/Earth Benefits: The goal of this project is to address the existence of a sensory and neuronal-based mechanism by which microgravity conditions provoke bone loss, using genetic and pharmacological in vivo approaches, with the long-term goal to propose novel and more targeted pharmacological avenues to prevent microgravity-associated bone loss. Our results, beyond contributing to our understanding of the mechanisms whereby bone homeostasis is controlled, have potential important clinical implications. One is that beta-2 AR pharmacological blockade may be able to counteract the bone loss associated with unloading conditions during bed rest or long-term space travel. Another implication is that patients with vestibular pathologies, especially bilateral dysfunctions, may present with low bone mineral density and be at higher risk for fracture than the general population. This may also be relevant to the association between vestibular dysfunctions and bone loss observed in aging individuals. Therefore, one could also speculate that the progression of bone loss during aging could be accelerated upon vestibular dysfunction. All these implications remain at the present time speculative but warrant further experimental and clinical investigations.

Task Progress & Bibliography Information FY2013 
Task Progress: SPECIFIC AIM 1: Determine if vestibular lesion causes bone loss by activation of the sympathetic nervous system. The first step of this study consisted in evaluating the effect of a bilateral vestibular lesion (VBX) on femoral bone mass. Using micro-ct analyses on 2-month old mice, we demonstrated that VBX induces a significant decrease in femoral bone mass one month after the lesions. Histomorphometric analyses are ongoing. The second step was to determine whether the sympathetic nervous system (SNS) mediates this VBX-induced bone loss. Using 2-month old beta-2 adrenergic receptors KO mice, we detected no bone change in BV/TV 1 month after VBX. Moreover, daily propranolol treatment had no effect on WT-sham mice but completely blunted VBX-induced bone loss in WT-VBX mice. Taken together, these results suggest that the SNS might be involved in VBX-induced bone loss. We are currently breeding a conditional beta-2 adrenergic receptors KO model lacking these receptors specifically on osteoblasts. We thus will be able to make the distinction between the central and peripheric SNS involvement in VBX-induced bone loss.

SPECIFIC AIM 2: Analyze the bone phenotype of mice devoid of vestibular gravity sensor. We used 3-month old Het–/– mice (Nox3het/J), lacking otoliths (gravity sensors), in order to mimic the decrease in vestibular stimulation in space. Micro-CT analyses revealed no bone changes in these mice compared to WT. Because of the lack of otoliths, we assumed that these mice should present a decrease in vestibular inputs and bone loss. However, Nox3 encodes a NADPH oxidase in hair cells which is involved in the downregulation of nitric oxide (NO) availability. Knowing that NO level in vestibular hair cells modulates the vestibular signals and that it is an important neuromediator/neuromodulator of the vestibular response, a constitutive high NO level in hair cells might explain our results in this model (aim 3 will help to clarify this).

SPECIFIC AIM 3: Test nitric oxide involvement in vestibular-related bone loss The Sod3flox/flox mice are currently at early stages of breeding. Preliminary experiments to define the titer of cre-adenovirus needed to inactivate Sod3 specifically in the inner ear are ongoing, using Rosa26 reporter mice. Successful vestibular histological sections have been obtained, which will be critical to measure the extent and specificity of cre-recombination in this model.

Bibliography Type: Description: (Last Updated: 04/12/2016)  Show Cumulative Bibliography Listing
 
 None in FY 2013
Project Title:  Contribution of the Vestibular and Sympathetic Nervous Systems to Space-Induced Bone Loss (Postdoctoral Fellowship) Reduce
Fiscal Year: FY 2012 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 11/01/2011  
End Date: 10/31/2013  
Task Last Updated: 10/26/2011 
Download report in PDF pdf
Principal Investigator/Affiliation:   Vignaux, Guillaume F. Ph.D. / Vanderbilt University 
Address:  Center for Bone Biology 
1211 Medical Center Drive 
Nashville , TN 37232 
Email: guillaume.f.vignaux@vanderbilt.edu 
Phone: 615-322-7883  
Congressional District:
Web:  
Organization Type: UNIVERSITY 
Organization Name: Vanderbilt University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Elefteriou, Florent  MENTOR/ Vanderbilt University 
Project Information: Grant/Contract No. NCC 9-58-PF02603 
Responsible Center: NSBRI 
Grant Monitor:  
Center Contact:   
Solicitation / Funding Source: 2011 NSBRI-RFA-11-01 Postdoctoral Fellowships 
Grant/Contract No.: NCC 9-58-PF02603 
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: POSTDOCTORAL FELLOWSHIP

A milestone was reached with the first step on the Moon, but the upcoming project to reach Mars poses additional challenges. DXA measurements realized after four- to-six-month space missions reported a 1-2 percent monthly decline in bone mineral density (BMD). BMD loss will certainly progress further following the six-month period required to reach Mars, and it is unlikely that fractional gravity on Mars will mitigate the bone loss that will occur during travel.

The vestibular system is disturbed in microgravity, inducing cardiovascular alterations via sympathetic activation. Sympathetic activation also provokes bone loss following stimulation of the b2-adrenergic receptor (b2AR) in osteoblasts -- the bone forming cells.

Hypothesis. Based on these observations, the researchers hypothesize that the vestibular system participates to the maintenance of BMD on Earth and its dysfunction under microgravity may contribute to the bone loss associated with space travel.

Preliminary findings have provided evidence to support the hypothesis. Vestibular lesion (VBX) using sodium arsanilate injections in rats destroyed bladderwort sensorial cells (gravity sensors) without any other neuronal damage and led to significant bone loss associated to a decrease in osteoblasts number. The researchers have observed a similar bone phenotype in mice after VBX.

Specific Aims

1) To determine if VBX causes bone loss by activation of the sympathetic nervous system in the VBX model using b-blocker-treated mice and mice lacking the b2AR globally or specifically in osteoblasts;

2) To analyze the bone phenotype of mice devoid of vestibular gravity sensor (Het-/- mice); and

3) To determine if vestibular stimulation by centrifugation could be used as a countermeasure to bone loss under microgravity.

This study may uncover of a new pathway of bone regulation, a novel approach for the treatment of low bone mass diseases on Earth, and novel countermeasures to reduce risk of bone fracture in microgravity.

Research Impact/Earth Benefits: This study may uncover of a new pathway of bone regulation, a novel approach for the treatment of low bone mass diseases on Earth, and novel countermeasures to reduce risk of bone fracture in microgravity.

Task Progress & Bibliography Information FY2012 
Task Progress: New project for FY2012.

Bibliography Type: Description: (Last Updated: 04/12/2016)  Show Cumulative Bibliography Listing
 
 None in FY 2012