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Keywords:

  • VESTIBULAR LESION;
  • BONE;
  • SYMPATHETIC NERVOUS SYSTEM;
  • MICROGRAVITY

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

Bone remodeling allows the conservation of normal bone mass despite constant changes in internal and external environments. The adaptation of the skeleton to these various stimuli leads credence to the notion that bone remodeling is a true homeostatic function, and as such is under the control of specific centers in the central nervous system (CNS). Hypothalamic and brainstem centers, as well as the sympathetic nervous system (SNS), have been identified as regulators of bone remodeling. However, the nature of the afferent CNS stimuli that may modulate CNS centers involved in the control of bone remodeling, with the exception of leptin, remains unclear. Based on the partial efficacy of exercise and mechanical stimulation regimens to prevent microgravity-induced bone loss and the known alterations in vestibular functions associated with space flights, we hypothesized that inner ear vestibular signals may contribute to the regulation of bone remodeling. Using an established model of bilateral vestibular lesions and microtomographic and histomorphometric bone analyses, we show here 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, as observed in rats under microgravity conditions. 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 β-blocker propranolol. Collectively, these data suggest that the homeostatic process of bone remodeling has a vestibulosympathetic regulatory component and that vestibular system pathologies might be accompanied by bone fragility. © 2013 American Society for Bone and Mineral Research.


Introduction

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

Skeletal integrity in adult mammals is maintained by the interplay between bone formation and bone resorption through the process of bone remodeling. This process involves osteoclasts—the bone-resorbing cells—and osteoblasts—the bone forming cells—and requires balanced activity between those two cell types to conserve normal bone mass in adults.

The internal milieu has a pronounced influence on bone cells, via both hormonal and neuronal signals. The presence of sympathetic nerves in the bone environment was reported more than 50 years ago[1, 2] and we have more recently shown, using genetic and pharmacologic approaches, that sympathetic nerves contribute to the regulation of bone homeostasis.[3] Osteoblasts express the beta-2 adrenergic receptor (β2AR) and respond to catecholamines or pharmacological βAR agonists with decreased proliferation[4] and induction of receptor activator of NF-κB ligand (RANKL) expression, which leads to enhanced osteoclast formation and bone resorption.[5, 6] Propranolol, a β1/β2AR nonselective antagonist, inhibits these effects of sympathetic activation and protects from ovariectomy-induced and unloading-induced bone loss in mice and rats,[5, 7-9]; in several retrospective studies, the use of β-blockade was associated with increased bone mineral density (BMD) and decreased fracture rate in humans.[3, 10, 11]

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.[12-16] Osteocytes are sensors of these stimulations and respond to them via mechanisms leading to enhanced bone formation.[17-21] In contrast, mechanical unloading as a consequence of bed rest or microgravity conditions causes bone loss in humans[22] and rodents,[23] 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.[24] The otolith system, which is part of the vestibular system in the inner ear, is symmetrically located in an isolated area of the petrous bone in the skull and 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[25-28] and in humans[29, 30] is well-documented and supported by anatomical projections from vestibular nuclei to autonomic centers of the brainstem.[31, 32] Vestibular inputs contribute to changes in sympathetic nerve outflow during movements and postural changes that follow stimulation of the vestibulosympathetic reflex,[33-36] as demonstrated by the orthostatic hypotension observed in cats with bilateral vestibular destruction.[37]

This body of knowledge and the coexistence of bone loss with changes in vestibular signals and sympathetic outflow, known to occur in microgravity conditions, led us to hypothesize that the vestibular system may contribute to the regulation of bone remodeling, in addition to the well-accepted direct contribution of altered mechanical loading to the skeleton. The existence of such a vestibular component, if demonstrated, could explain why daily muscular exercise programs are only partially effective in preserving bone mass under microgravity conditions,[38-41] and would be consistent with the sympathetic and cardiovascular changes observed in microgravity.[42, 43] We addressed this hypothesis by analyzing the skeletal response of rats to a validated model of bilateral vestibular lesions induced by arsanilate treatment.[44]

Subjects and Methods

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

General procedures

Animal experiments were carried out in accordance with European Communities Council Directive 86/6609/EEC as well as French law. Sprague Dawley rats (Janvier, Saint Berthevin, France) were group-housed (2–3/cage) under conditions of constant temperature (21°C ± 1°C) and humidity, with a 24-hour light-dark cycle (lights on 8:00 a.m. to 8:00 p.m.) and food and water freely and continuously available. Chow normal diet contained 0.73% calcium, 0.52% phosphorus, and 600 UI of vitamin D3/kg. Weight, water consumption, and food consumption were measured weekly. Rats were injected with calcein (10 mg/kg) 2 and 6 days prior to euthanasia.

Drug administration

Propranolol (Sigma-Aldrich; 0.5 g/L), a beta-2 adrenergic receptor antagonist, was given ad libitum in drinking water.

Bilateral vestibular lesion procedure and vestibular syndrome clinical scale

At the beginning of the study, 3-month-old female rats were anesthetized in an isoflurane chamber at 3.5% oxygen (flow rate 2 L/min) and maintained under a nosecone at 2% oxygen (flow rate 0.8 L/min). Each rat received a bilateral transtympanic injection of 0.1 mL sodium arsanilate solution (D-Arsenilic; Sigma-Aldrich) at a dose of 50 mg/mL (diluted in saline solution at 0.9%). Sham rats received bilateral transtympanic injections of 0.1 mL of saline solution. These doses induce peripheral labyrinthectomy without damaging the VIII cranial nerves.[44]

The behavioral effects of chemical vestibular lesions were evaluated with a previously validated clinical vestibular scale.[45] This scale assesses six static or dynamic locomotor points, with a value ranging from 0 to 4 for each criterion. Three categories of spontaneous motor behavior were evaluated, including circular walking, retropulsive movement, and abnormal head bobbing, alongside three reflex behaviors, including tail-hang, contact inhibition of righting, and air righting. Clinical score was recorded for each rat at 1, 2, 3, 7, 15, 23, and 30 days following vestibular lesion induction. The final score for each rat was given as a percentage of the maximum scale value of 24. Bobbing is an abnormal head movement characterized by the periodic backward extension of the neck, circular walking is circular locomotor activity, and rearing is defined as retropulsive movement (backward walking of the animal). When the animal is lifted from the table, a successful tail-hang reflex results in the forelimbs reaching out in anticipation of contacting a surface; rats with vestibular lesion induction (VBX) often bend themselves ventrally and occasionally attempt to climb their own tails, which can lead them to fall on the back of their skulls. Contact inhibition related to the righting reflex reflects the ability and speed to go from a supine to a prone position on a table. The rat is turned over into a supine position with a grid placed to touch its feet while its back maintains contact with the table. Sham rats turn over to the physiological prone position while VBX rats stay in a supine position, attempting to walk with their feet upon the grid. The air-righting reflex is tested by holding animals supine and dropping them onto a padded cushion from a height of 30 cm. Sham rats are able to right themselves, while BVL rats fail to right and land on their backs.

Horizontal locomotor activity

A first group of 16 three-month-old female rats (8 sham, 8 VBX) was anesthetized. A cutaneous incision was performed in the umbilical area, muscle planes were spread back and a PA-C40 telemetric captor (DSI, St. Paul, MN, USA) was fixed in the abdominal cavity of each rat with a nonabsorbable suture (Surgipro 3/0). The cutaneous incision was then closed with absorbable sutures (Surgipro 4/0). After 1 week of rest, the animals received a bilateral transtympanic injection of either arsanilate or saline solution and were placed in individual cages in a dedicated room. Under each cage a transmitter plate continuously received the captor signal that was recorded on a computer. Analysis of the recorded horizontal locomotor activity was performed by range from day –3 to 0, day 0 to 4, and day 30 to 33 using the Dataquest A.R.T. software (DSI).

Locomotion topography was recorded on another group of 16 three-month-old female rats (8 sham, 8 VBX). One month after receiving bilateral transtympanic injection of arsanilate or saline solution, each rat was placed in an open field for 8 minutes. An infrared floor was placed underneath the field and a camera recorded each animal's horizontal movements. The locomotion topography was analyzed using a VideoTrack (ViewPoint Life Sciences, USA) system to obtain the time and speed of locomotion.

Densitometric study

BMD (g/cm2) was measured at baseline in 3-month-old female rats and 1 month following lesion induction. Acquisitions were performed with dual-energy X-ray absorptiometry (DXA) with a Hologic QDR-4500A (Waltham, MA, USA) on anesthetized animals, using ultrahigh-resolution (0.307 mm per line for femoral and lower spine acquisitions) and high-resolution (1.512 mm per line for whole body [WB]). WB included the entire rat skeleton. Each animal was placed in a ventral position on a thin Plexiglas plate under anesthesia. Rats were stretched out to avoid a scoliotic position and the back legs were externally rotated and fixed in place with adhesive tape. The sequence of acquisition for each animal progressed from WB to homolateral and contralateral whole femur, distal femoral metaphysis (DFM), and diaphysis (FD), and then the third and fourth lumbar vertebrae (L3 and L4). The right and left BMD of the whole femur, DFM, and FD were then respectively averaged. Lower spine BMD was defined as the average of L3 and L4. Bone loss was expressed in percentage of sham BMD.

Micro–computed tomography analysis

The right femur and lumbar vertebrae (L3 and L4) from each animal were dissected and fixed overnight in 4% phosphate-buffered formalin and then transferred to 70% ethanol, loaded into 12.3-mm-diameter scanning tubes, and imaged using micro–computed tomography (µCT) (µCT40; Scanco Medical, Bassersdorf, Switzerland), according to the JBMR guidelines for µCT analysis of rodent specimens.[46] The scans were integrated into three-dimensional (3D) voxel images. A Gaussian filter (sigma = 0.5, support = 2) was used to reduce signal noise, and a threshold of 400 was applied to all analyzed scans. Scans were done at 12-µm resolution (E = 55 kVp, I = 145 µA). For femurs, 200 transverse slices of the distal femur were taken of the region extending from the epicondyles toward the proximal ends of the femurs. Manual analysis excluded cortical bone and primary spongiosa from the analysis of cancellous bone tissues. All trabecular measurements were made by manually drawing contours every 10 to 20 slices and using voxel counting and sphere-filling distance transformation indices.

Histomorphometry

The right femur from each animal was fixed in 4% phosphate buffered formalin, dehydrated, and embedded, undecalcified, in methyl methacrylate using standard procedures. Longitudinal sections were prepared using a Leica RM2255 microtome (Leica Microsystems, Heerbrugg, Switzerland). Histomorphometry measurements were performed using the Bioquant Image Analysis System (R&M Biometrics, Nashville, TN, USA). Distal femur cancellous bone measurements (osteoblast surface per bone surface [Ob.S/BS], osteoclast surface per bone surface [Oc.S/BS], trabecular number [Tb.N], trabecular thickness [Tb.Th], trabecular separation [Tb.Sp]) were restricted to the secondary spongiosa.

Gene expression

Total RNA was extracted using TRIzol (Invitrogen, Grand Island, NY, USA) and cDNAs were synthesized following DNase I treatment using the high-capacity cDNA reverse-transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative PCR (qPCR) was performed using TaqMan gene expression assays. The probe and primer sets for Hprt (Rn01527840_m1) and Ucp1 (Rn00562126_m1) were obtained from Applied Biosystems.

Statistics

All data are presented as means ± SD. Statistical analyses were performed using one-way ANOVA for multiple comparisons followed by post hoc pairwise comparison with Tukey adjustment and unpaired two-tailed Student's t tests for two-group comparisons. For all analyses, p < 0.05 was considered significant.

Results

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

Rat vestibular lesions trigger a strong vestibular syndrome

In order to determine whether vestibular signals influence bone homeostasis, the model of arsanilate vestibular lesions in rats was selected. This type of lesion induces the formation of large vacuoles below the sensory cell layer of the inner ear, near the synaptic contacts, between the sensory cells and the neuronal processes that arise from Scarpa's ganglion neurons and the swellings of the calyx and bouton nerve terminals. As reported,[44] the lesion is restricted to the vestibular sensory organs, because no diffusion of arsanilate into the external ear, Eustachian tubes, or through the eighth cranial nerve sheath leading to the brainstem was observed. In this new study, lesions were generated bilaterally in 3-month-old female rats using a dose of 5 mg of arsanilate per ear, as described.[44] Following lesion induction, the successful generation of a vestibular syndrome in arsanilate-treated rats was first assessed using a scale based on the measurement of six different static or dynamic spontaneous locomotor behaviors not expressed in sham animals[45] (see Subjects and Methods). The global score of vestibular syndrome ( = 0 in sham rats) increased during the first 3 days in arsanilate-treated rats and slightly decreased, but remained significantly different from sham rats during the entire study period (30 days) in 100% of arsanilate-treated rats (Table 1). These results validate the effectiveness and confined nature of the arsanilate vestibular lesions generated in these animals.

Table 1. Clinical Vestibular Syndrome Scores in VBX Rats
Time post-VBXCyclingRearingBobbingTail-hanging reflexContact inhibition reflexAir righting reflexGlobal score
  1. Values for scores are mean ± SD (n = 10/group); all values are significantly different than for sham rats, whose scores are null for each parameter.

  2. VBX = vestibular lesion.

1 day4.0 ± 0.03.3 ± 0.54.0 ± 0.04.0 ± 0.04.0 ± 0.02.8 ± 1.022.0 ± 0.8
2 days4.0 ± 0.03.3 ± 0.54.0 ± 0.04.0 ± 0.04.0 ± 0.03.3 ± 1.022.5 ± 1.0
3 days4.0 ± 0.03.5 ± 0.64.0 ± 0.04.0 ± 0.04.0 ± 0.03.3 ± 1.022.8 ± 0.5
7 days4.0 ± 0.01.8 ± 1.34.0 ± 0.04.0 ± 0.04.0 ± 0.03.0 ± 1.220.8 ± 1.9
15 days4.0 ± 0.01.3 ± 1.54.0 ± 0.04.0 ± 0.04.0 ± 0.03.0 ± 1.220.3 ± 1.7
23 days3.8 ± 0.51.0 ± 2.04.0 ± 0.04.0 ± 0.04.0 ± 0.02.8 ± 1.019.3 ± 1.0
30 days3.0 ± 2.01.0 ± 2.04.0 ± 0.04.0 ± 0.04.0 ± 0.02.8 ± 1.018.9 ± 1.5

Vestibular lesions lead to a low bone mass phenotype without affecting metabolic homeostasis

One month following VBX, rats were euthanized, lumbar and femoral bones were collected, and BMD was measured by high-resolution DXA on whole-body, whole-femur, distal femoral metaphysis, femoral diaphysis, and vertebrae (Fig. 1A). At this time, no significant changes in whole-body and vertebrae BMD could be observed by DXA (Fig. 1B). A significantly reduced BMD was, however, detected on the whole femur (–4.0% versus sham), the femoral distal metaphysis (–11.3% versus sham), and diaphysis (–5.2% versus sham).

image

Figure 1. (A) Densitometric area measurements. (B) Whole body, vertebral, and femoral bone mineral density (BMD) in VBX rats 1 month after lesion (n = 10; error bars are SDs; ***p < 0.005 versus sham). DFM = femoral metaphysis; FD = femoral diaphysis.

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DXA and intake measurements were also used to assess additional metabolic parameters that could possibly influence bone homeostasis. Body weight, lean and fat mass, and food and water intake were not different between sham and VBX rats at the time of euthanasia (Table 2). These results suggest that vestibular signals contribute to bone homeostasis, independently of major metabolic changes, and that BMD in lower limbs is to a greater extent under this type of vestibular control.

Table 2. Metabolic and Nutrition Parameters in Sham and VBX Rats (n = 10/group)
ParameterGroup0 days15 days30 days
  1. Values are mean ± SD.

  2. Sham = control; VBX = vestibular lesion.

Weight (g)Sham302.2 ± 5.9314.8 ± 6.0323.3 ± 6.4
 VBX296.6 ± 6.1304.2 ± 17.0331.7 ± 9.8
Food intake (g/d)Sham33.7 ± 1.133.6 ± 1.333.5 ± 2.0
 VBX36.1 ± 0.335.3 ± 0.534.2 ± 0.8
Drink (mL/d)Sham19.2 ± 0.420.4 ± 0.621.0 ± 1.0
 VBX16.6 ± 0.318.2 ± 0.319.0 ± 0.4
Lean mass (g)Sham256.7 ± 11.6268.7 ± 13.3292.9 ± 20.1
 VBX250.9 ± 12.3270.1 ± 16.0294.1 ± 27.9
Fat mass (g)Sham46.4 ± 6.850.2 ± 7.854.9 ± 8.7
 VBX46.7 ± 14.552.6 ± 13.258.4 ± 10.6
Fat mass percentage (%)Sham15.3 ± 1.915.4 ± 2.115.8 ± 2.4
 VBX15.5 ± 3.815.9 ± 3.016.5 ± 2.2

Vestibular lesions provoke bone loss despite increased locomotor activity

Low locomotor activity can be associated with a reduction in BMD.[47] Because disorientation is observed in microgravity conditions in humans, we asked whether the bone loss observed in VBX rats may be secondary to disorientation and altered locomotor activity, using video-tracking and telemetry (DSI system) in sham and VBX rats. Video-tracking of rats placed in an open field at day 30 postlesion revealed that VBX rats explored the entire surface of the open field, whereas sham rats preferentially remained in the peripheral area (Fig. 2). Moreover, VBX rats displayed disorganized exploration with spontaneous circular walking and rearing episodes, whereas sham rats moved in a straight line.

image

Figure 2. Outlines of horizontal locomotor activity evaluated by video-tracking in rats 1 month after sham operation or VBX (n = 8/group).

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Telemetric recording 24 hours per day to record continuous locomotor activity revealed no significant difference between sham and VBX rats during the 3 days prior to lesions or during the 4 days following vestibular lesions. Unexpectedly, 1 month after vestibular lesion, locomotor activity in the VBX group was in fact approximately 2 times higher compared to the sham group (Fig. 3A, B). This increase in locomotor activity was detected during both rest (8:00 a.m. to 8:00 p.m.) and wake (8:00 p.m. to 8:00 a.m.) periods (Fig. 3B). Twitching or spasms were not observed in VBX rats, and the increase in locomotor activity was associated with actual movement rather than random nonspecific motion. Although the etiology underlying this increase in locomotor activity remains unknown, these observations refute the hypothesis that vestibular lesions may cause bone loss via a decrease in bone strain subsequent to reduced locomotor activity.

image

Figure 3. Horizontal locomotor activity evaluated by telemetric measurement in rats after sham operation or VBX. (A) Outlines of horizontal locomotor activity before VBX (day –3 to 0, top panel), following VBX (day 0–4, middle panel), and before euthanasia (day 30–33, bottom panel). (B) Horizontal locomotor activity in relation to circadian rhythm (n = 8/group; error bars are SDs; *p < 0.05 versus sham).

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Beta-adrenergic receptor blockade by propranolol prevents the inhibitory effect of vestibular lesions on bone mass

The vestibular system has sympathetic nervous system projections[48] and sympathetic activation induces bone loss.[6] The observation that vestibular lesions in rats induce a low bone mass phenotype thus led us to hypothesize that the vestibular system may regulate bone remodeling via activation of sympathetic nerves. To experimentally address this question, we used propranolol, a β1/β2AR nonselective blocker, to inhibit the response to sympathetic outflow putatively resulting from vestibular lesion. Sham and VBX rats were treated with propranolol for 1 month following vestibular lesion and then bone parameters were assessed in femurs and lumbar vertebrae using -D µCT. Our µCT analyses confirmed a significant reduction in femoral bone volume/total volume (BV/TV) in VBX rats compared to sham controls (0.27% in VBX versus 0.39% in sham) and a highly significant decrease in trabecular number and thickness, accompanied by an increase in trabecular space (Fig. 4A). As observed using DXA measurements, no changes in bone parameters were detectable in the axial skeleton (Supplemental Fig. 1). Propranolol treatment had no significant effect on femoral BV/TV in the sham group, did not alter food and water intake, nor body weight and locomotor activity, but blunted bone loss and all other changes in bone induced by VBX, including BV/TV, Tb.Sp, Tb.Th, and Tb.N (Fig. 4A).

image

Figure 4. (A) Effects of propranolol treatment on bone microarchitecture parameters 1 month after sham operation or VBX (µCT measurements). (B) Histomorphometric analyses of osteoblast surface/bone surface (Ob.S/BS) and osteoclast surface/bone surface (Oc.S/BS) (n = 10/group; error bars are SDs; *p < 0.05, ***p < 0.005). (C) Ucp1 expression in brown adipose tissue 1 month after sham operation or VBX (n = 6/group; **p < 0.01). BV/TV = bone volume/total volume; Tb.N = trabecular number; Tb.Th = trabecular thickness; Tb.Sp = trabecular separation.

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Further histomorphometric analyses on undecalcified sections from femoral bones were performed to examine the alterations in cellular parameters following vestibular lesion and propranolol treatment. A significant decrease in Ob.S/BS was observed in VBX rats, as observed following βAR stimulation by isoproterenol in mice; however, no significant change in the bone formation rate (BFR/BS) could be detected at this time point (data not shown). Oc.S/BS was not affected by vestibular lesions (Fig. 4B). As observed for bone structural parameters, propranolol treatment did not significantly increase Ob.S/BS in sham rats but blunted the reduction in Ob.S/BS observed in VBX rats, whereas it had no detectable effect on Oc.S/BS. Altogether, these results suggest that vestibular lesions induce bone loss, at least in part, via sympathetic activation. This conclusion is further supported by an increase in Ucp1 expression (a marker of sympathetic outflow) detected in the brown adipose tissue of VBX rats compared to sham controls (Fig. 4C).

Discussion

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

Bone remodeling is under the control of hormonal and paracrine/autocrine factors that maintain the balance between bone formation by osteoblasts and bone resorption by osteoclasts, in turn allowing the preservation of optimal serum mineral homeostasis, bone structure, and bone quality. Neuronal centers in the central nervous system (CNS) contribute to the homeostatic regulation of bone remodeling, and the sympathetic nervous system constitutes one of the efferent pathways whereby these centers communicate with bone cells. In this study, we provide evidence that signals from the inner ear vestibular system are required for normal bone homeostasis, and that this function may be mediated via sympathetic nervous system efferences from central vegetative centers. These findings have possible clinical implications for patients with a history of vestibular deficits such as labyrinthectomy, antibiotic treatment (aminoglycosides and platinum-based chemotherapy), vestibular neuritis, Ménière's disease, and possibly for long-term space travel endeavors in microgravity conditions.

Bed rest and microgravity conditions both lead to bone loss. Mechanical unloading partly explains this effect, because osteocytes embedded in cortical weight-bearing bones normally sense shifts in fluids caused by loading and trigger an anabolic reaction in response to it. The limited effect of resistance exercise regimens that partly restore mechanical stimulation to bone during unloading or microgravity conditions, however, suggested the existence of an additional mechanism(s) involved. The bone loss induced in our rat model of arsanilate-based vestibular lesions supports the hypothesis that a functional vestibular system is required for the conservation of normal bone mass, and suggests that this system may influence the adaptation of bone to ambient gravity and weight. The vestibular system appeared more than 500 million years ago[49] and remains the principal sensory organ perceiving and encoding gravity on Earth, acting primarily through the otoconia of macular hair cells in every mammalian species; the notion that vestibular signals could integrate with and regulate major homeostatic functions, including cardiovascular function and the regulation of bone remodeling, is thus not totally surprising.

Of interest is that the bone loss observed in VBX rats is restricted to load-bearing bones, which are also the main skeletal sites of bone loss in microgravity conditions, and a result of reduced bone formation rather than increased bone resorption, as observed in rats following spaceflight conditions.[38, 50-58] If sympathetic activation is a consequence of the bone loss induced by vestibular lesions, one can hypothesize that lower extremities and load-bearing bones may be differentially innervated, receive different sympathetic signals, or respond to them differently compared to bones of the axial skeleton; however, this hypothesis remains to be addressed.

The contribution of the sympathetic nervous system to VBX-induced bone loss is supported by the increase in brown adipose tissue Ucp1 expression observed in VBX rats and by the protective effect of propranolol on VBX-induced bone loss. It is also consistent with data indicating that vestibular stimulation leads to a decrease in sympathetic nervous system (SNS) activity in hindlimbs.[59] However, it cannot be excluded that other peripheral mediators are involved. It is noteworthy that the lack of changes in body weight, muscle mass, and food intake in VBX rats does not support chronic activation of the hypothalamic-pituitary-adrenal axis as a mechanism whereby vestibular lesions would cause bone loss. This conclusion is also supported by the corticosteroid-independent bone loss observed in rats during spaceflight.[57] In addition, the precise inner ear structures, neurons, and neuromediators, as well as the CNS circuitry involved in this newly identified bone vestibulosympathetic regulatory system, remain unknown. On one hand, electrophysiological studies indicated the existence of a predominant indirect pathway through the lateral medullary reticular formation between the vestibular nucleus (VN) and the rostro-ventro-lateral medullary structure (RVLM), the latter being defined as the main vegetative center.[33, 60] On the other hand, neuroanatomical studies reported an indirect and clear connection between the VN and the RVLM through the tractus solitary nucleus,[32] with the RVLM being then directly connected to the sympathetic medullary tractus innervating arterioles and possibly bone tissues.[61] Measurement of peripheral sympathetic tone induced by vestibular stimulation along the abdominal cavity supported a distinct and independent modulation depending on the targeted organ.[33, 35] Interestingly, the vestibular-related sympathetic response presented a somatotopy in vascular responses, predominantly focused on lower limbs in cats,[62, 63] similar to the lower limb specificity of the vestibular lesion effect on the skeletal system reported in our study. This suggested the possibility that bone loss induced by vestibular lesions could be mediated by decreased perfusion of the limbs, which is known to occur in microgravity conditions.[42, 64, 65] In cats, however, inadequate vascular regulation of lower limbs caused by vestibular lesions recovered progressively within 1 week,[66] suggesting that reduced bone tissue perfusion may not be a predominant cause of the bone loss resulting from VBX.

The relative contribution of vestibular and mechanical signals to bone loss in unloading/microgravity conditions remains unclear. The cardiovascular effects induced by vestibular alterations in feline experimental models or during microgravity conditions, for instance, are relatively variable (from 1 week for blood pressure in bilabyrinthectomized cats up to 6 months for heart rate and mean arterial pressure in astronauts).[66, 67] It thus remains unknown whether the effect of vestibular lesions on bone remodeling is similarly transient (i.e., compensated for in time), or prolonged. This question may be relevant to the evolution of bone loss during long-term space travels and to the reduced ability of astronauts to recover BMD following their return to Earth.[68] Assessing the possible occurrence of compensatory mechanisms several months after vestibular lesion induction in our rat model, in loading or unloading conditions, will be required to address this question. This question may also be applicable to the absence of alterations in BFR and Oc.N observed 4 weeks following vestibular lesion induction in rats, which does not recapitulate what was observed in mice upon daily adrenergic stimulation via sympathetic agonists for 6 weeks. Additional study time points will be necessary to determine whether such differences are caused by the time frame of the experiment, by the existence of SNS-independent mechanisms, or by distinct effects of chronic endogenous sympathetic activation versus intermittent exogenous pharmacological βAR agonists on bone remodeling.

These findings, beyond contributing to our understanding of the mechanisms whereby bone homeostasis is controlled, have clinical implications. One is that βAR pharmacological blockade may be able to counteract the bone loss associated with long-term space travel. It is important to emphasize that bone unloading, nutritional deficiencies,[41] decreased calcium intestinal absorption, as well as changes in blood volume distribution and psychological factors may contribute to bone loss. If vestibular dysfunction and psychosocial factors typical of confined and microgravity conditions combine to activate sympathetic outflow during space travel, one could speculate that sympathetic blockade, along with corrected energy intake and vitamin D status, may be beneficial. Another implication is that patients with vestibular pathologies, especially bilateral dysfunctions, may present with low BMD and be at higher risk for fracture than the general population. Vestibular dysfunctions are also more prevalent 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.

Acknowledgments

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

This work was supported by grants from the Centre National d'Etudes Spatiales (CNES, CNES-2012-004884), from ANR-09-BLAN-0148-07, from Lower-Normandy Region, France, and from the National Space Biomedical Research Institute through NASA NCC 9-58. We thank Dr. D. Perrien (VCBB) for his technical expertise with µCT and for critical reading of the manuscript, Sameena K. Campbell for editing the manuscript, Pr. P. Mulder (INSERM U1096, Rouen) for the telemetric system supply, and Dr. JP. Sabatier (CHU of Caen) for his technical expertise with DXA.

Authors' roles: Study design: VG, BS, DP, and EF. Study conduct: VG, NJ, and PB. Data collection: VG and NJ. Data analysis: VG and EF. Data interpretation: VG, BS, and EF. Drafting manuscript: VG, BS, PB, and EF. Revising manuscript content: VG, BS, PB, and EF. Approving final version of manuscript: VG, BS, DP, and EF take responsibility for the integrity of the data analysis.

References

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  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
jbmr1940-0001-sm-SupplFigS1.tif651KSupplementary Figure S1
jbmr1940-sm-0002-SupplFigLegend.doc35KSupplementary Figure Legend

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