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.

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.

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.

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.

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.