Botulinum toxin (A)-induced bone loss is associated with increased blood velocity and reduced vascular bone porosity

Abstract

Disuse-induced bone loss is a common consequence of spaceflight and prolonged bed rest. Intraosseous blood vessel volume and number are decreased in rodents after sciatic nerve resection, and femoral and tibial perfusion and blood flow to the femoral shaft and marrow are reduced after hindlimb unloading. However, it is unclear if alterations in the flow of blood contribute to botulinum toxin (BTX)-induced bone loss. The objective of this study was to assess patterns of tibial bone loss and alterations in blood flow in murine hindlimbs following BTX injection. We hypothesize that flow of blood to the affected hindlimb will diminish along with bone mass and structure. Skeletally mature C57Bl/6J female were injected with BTX (n = 15) or vehicle (n = 14). Paralysis was confirmed using digit abduction, wire hang tests, and activity analysis. In vivo microCT and ex vivo synchrotron tomography were used to assess bone mass, microstructure, (re)modeling, as well as vascular and lacunar porosity. Blood flow in the hindlimbs and cardiac structure/function was monitored by echocardiography. After 3 wk, BTX-injected tibiae had 16% lower cortical thickness and 66% lower trabecular bone volume fraction compared to baseline. MicroCT-based timelapse morphometry showed bone loss was predominantly at endocortical surfaces. Bone loss in the contralateral limb was coincident with reduced rearing capability of BTX-injected mice compared to vehicle controls. Bony vascular canal thickness and surface area were reduced, but there was no change in lacunar properties due to BTX. In vivo ultrasound demonstrated increased velocity time integral for blood flow due to BTX injection in femoral and popliteal but not in saphenous arteries. Thus, BTX led to significant bone loss in hindlimbs, while increasing blood velocity in the femoral popliteal arteries and decreasing vascular porosity. The vascular response to BTX differs from what has been observed in other hindlimb unloading models.

Graphical Abstract

Graphical Abstract.

Introduction

Bone (re)modeling is a life-long process driven by the mechanical forces exerted on bone through gravity and muscular contractions via tendons and ligaments. In an altered gravitational environment such as in microgravity, bone loss occurs in the absence of these mechanical forces.^1,2 Other conditions involving disuse include paralysis and long-term bed rest, which also cause decreases in bone mineral density.³ Mechanical forces vital to bone homeostasis are sensed by osteocytes, which act as the primary mechanosensor in bone. Osteocytes—the most abundant cells in bone—are embedded in the bone tissue within lacunae. They orchestrate bone (re)modeling by signaling to bone-forming osteoblasts and bone-resorbing osteoclasts.⁴ It is well accepted that interstitial fluid flow in the vast osteocyte lacunar-canalicular network is essential for mechanosensing by osteocytes and for delivery of nutrients to bone cells driving the anabolic changes.⁵ Additionally, bones are highly vascularized with multiple routes of entry/exit for blood and a complex network of trans-cortical vessels that interface with the interstitial fluids of bone.⁶

Blood is delivered to the murine hindlimb via a main conduit femoral artery, which bifurcates proximal to the knee into saphenous artery, supplying superficial musculature and tissue of the medial hindlimb, and popliteal artery, supplying the tibia and deeper tissues.⁷ Blood flow in the marrow space drives interstitial fluids into the osteocyte lacunar canalicular network.⁸ In the bone cortex, the blood vessels occupying the vascular porosity of bone serve as sites of exchange for nutrients, oxygen, and waste products with the surrounding osteocyte network.⁸ Thus, adequate perfusion and flow of blood is essential for bone (re)modeling and reduced vascularization is reported in populations affected by disuse osteopenia and aging.⁹

Multiple animal models that either remove ground reaction forces or eliminate muscular forces acting on bone seek to replicate the deteriorative musculoskeletal changes in disuse conditions. The most commonly used is the hindlimb suspension model, developed by Dr. Morey-Holton at NASA, which aims to mimic microgravity.¹⁰ Other disuse models include cast immobilization, chemical denervation using botulinum toxin A (BTX), and surgical denervation to mimic paralysis and prolonged bed rest.¹¹ Numerous studies have shown the degree of bone loss is variable across different models of disuse.¹² The advantage of using a BTX model is that it is non-invasive, well-controlled and provides an easy access for imaging the affected limb in vivo. Of interest, it was shown that bone loss in many models of unloading is associated with alterations in vascular parameters. Preclinical studies of simulated microgravity in rats¹³ and mice¹⁴ demonstrated reductions in blood flow and vascularity. Similarly, in mice with spinal cord injury and sciatic nerve resection bone loss was associated with decrease in intraosseous blood vessels.¹⁵ In contrast, in a surgical model of disuse, increase in blood flow was shown to precede bone loss.¹⁶ Although the BTX model has been used to examine bone loss, the changes in bone vascularity in this model have not been investigated. Prior studies demonstrated that BTX injection in the skin,¹⁷ perivascular¹⁸ or vascular¹⁹ regions leads to vasodilation and increased blood flow. Thus, alterations to vascular flow have been implicated as a potential contributing factor in disuse-induced bone loss, but whether this is also a factor in BTX models remains unclear.

Since the flow of blood drives bone interstitial fluid transport and pressure, and thus subsequent osteocyte mechanosensation, studies have tried to characterize osteocyte lacunar properties such as density, volume or porosity in models of muscle disuse. After space flight, significant decreases in lacunar volume were detected,²⁰ while in a hindlimb unloading model no changes in osteocyte lacunar density were found.²¹ Following BTX injection, both no difference in osteocyte lacunar volume²² and reduced osteoclast lacunar density²³ were reported. Thus, it remains unclear if unloading or BTX treatment are associated with changes in the osteocyte lacunar network.

In our study, we used a single BTX injection in the mouse hindlimb musculature to induce flaccid paralysis and quantified bone loss and blood flow and velocity. We hypothesized that the vasculature supplying the hindlimb and tibia would be diminished in response to BTX-induced disuse. Further, we hypothesized changes in bone structure and reduced osteocyte lacunar and vascular porosity in the tibia would develop. The objectives of the study were to assess patterns of BTX-induced changes in (1) bone morphology, (2) osteocyte lacunar properties, (3) blood flow and velocity in hindlimb arteries, and (4) morphology of blood vessels of the hindlimb and the intraosseous vascular porosity.

Materials and Methods

Animals

Animal use was approved by the McGill Animal Compliance Office, the Facility Animal Care Committees at the Shriner’s Hospital for Children (SHC) and Jewish General Hospital (JGH) (AUP#2020-8192). Twenty-six-week-old female C57BL/6J mice (Jackson Laboratories) were housed up to 5 animals/cage, with ad libitum food and water.

Experimental design

Twenty-nine animals were randomly assigned to BTX or vehicle group. Fourteen animals (BTX (7), Vehicle (7)) remained housed at SHC for activity testing performed before injection (day 0) and on days 2, 3, 4, 8, 9, 10, 11, 12, 16, 18, and 20 (Figure 1A). Fifteen animals (BTX (8), vehicle (7)) were allocated to investigate bone (micro-CT imaging, synchrotron vascular and lacunar network imaging) and vascular changes (ultrasonography, Microfil perfusion). Both tibiae were assessed by in vivo microCT at SHC 3 d before BTX injection. Animals were transported to the JGH and acclimatized for 48 hr. Echocardiography was performed on days 1, 9, and 19. Assessments of left and right hindlimb vascular properties were performed immediately before the injection into the left hindlimb muscles (day 0) and on days 2, 5, 7, 13, and 15 post-injection (Figure 1A). Mice were transported to SHC on day 21, in vivo microCT of both tibiae were collected, and mice were euthanized and perfused with the Microfil contrast agent (Flow Tek Inc.) via left ventricular perfusion. Whole hind limbs with the soft tissue intact were dissected and imaged using ex-vivo microCT. Tibia bones were dissected and preserved in 70% ethanol for imaging lacunar network properties.

Figure 1.

BTX-induced paralysis and changes in locomotion. (A) Experimental timeline denoting all procedures carried out over the 3-wk unloading period. (B) Paralysis score of BTX-injected animals. B, left: an example of the digit abduction test demonstrating lack of abduction in the BTX-injected (red circle - on the left) limb and normal response in the contralateral (green circle - on the right) limb, B, right: average combined scores for digit abduction and wire hanging in BTX-injected animals; all vehicle-injected animals scored 0. Paralysis scores in BTX-injected animals were significantly higher than vehicle-injected at all time points, ^*indicates significant difference of paralysis score from highest paralysis point (day 2), p < .05, (C) open field assessment of average distance traveled, active period, and rearing counts at baseline [B], at peak paralysis [PP, days 2-8], and at recovery period [R, day >8] in vehicle- (open circles) and BTX- (closed circles) injected mice. Data presented as means ± SD; ANOVA main effects: (a) treatment; (b) time period; (c) interaction, ^#p < .05; ^##p < .01 indicate significant difference based on Tukey–Kramer post-hoc test.

Botulinum toxin injections

BTX (Botox, Allergan) was diluted in saline (10 mL) to a concentration of 10 U/mL. On day 0, the BTX cohort were injected in the left limb with 2 U/100-g body weight divided equally between calf and quadricep muscle groups. The vehicle cohort received a saline injection in the left limb using the same protocol.²⁴ Following injection, animals were monitored for body weight and degree of paralysis using hindlimb abduction test²⁵ and wire hang test²⁶ daily during the first week, then bi-daily afterwards.

Activity monitoring

Animals were acclimated in the open field boxes with a mounted 3D-camera (Bioseb) (40 cm × 40 cm × 40 cm) for 1 hr/d for 2 wk. Data were collected at approximately the same time of the day (Figure 1A). Animals were allowed to roam for 30 min, then their movements were recorded for 15 min and data on total distance traveled, average speed, and rearing count were acquired.

In vivo microCT imaging

In vivo microCT imaging (Skyscan 1276, Bruker; 70 kVp, 57 μA, 0.3° rotation step, 0.5 mm Al filter) was used to collect 8 μm isometric voxel resolution images of tibiae at baseline (day 3) and before euthanasia (day 21). Animals were anesthetized with isoflurane (2% in 0.6 L/min O₂) during scans and kept immobile using a custom 3D-printed mouse bed. Both tibiae were captured, each scan took ~30 min. Tibia scans were 3D reconstructed using NRecon software (Bruker). The 2 volumes of interest were cortical bone in the tibial mid-diaphysis (extending 5% of the bone length at midline) and cortical and trabecular bone in the proximal tibial metaphysis (100 μm below the growth plate, extending 10% of the bone’s length). For the cortical bone of the mid-diaphysis region a global threshold was 601 mgHA/cm³, for the proximal tibial metaphysis—564.36 mgHA/cm³ for cortical and 348.29 mgHA/cm³ for trabeculae bone. Bone volume fraction (BV/TV), cortical thickness (Ct.Th), marrow area (Ma.Ar), cortical area (Ct. Ar), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were assessed using XamFlow software (V.1.8.8.0, Lucid Concepts AG) with a custom workflow following a prior CTAn protocol.²⁷ Results were validated across the 2 software programs using a separate dataset to ensure consistency.

MicroCT-based timelapse morphometry

The 2 volumes of interest acquired for the static microCT analysis were analyzed using timelapse histomorphometry. The day 21 images were registered using the Elastix image registration python library, integrated into the Xamflow software using previously defined and validated registration parameters.^28,29 Timelapse histomorphometry was performed using a previously developed MatLab script (https://github.com/BWillieLab/Timelapse-Morphometry), also integrated into XamFlow software,^28,29 to assess eroded (EV/BV, ES/BS) and mineralizing (MV/BV, MS/BS) volume and surface fractions over the 21-d period.

In vivo ultrasound imaging

A 40 MHz transducer (VEVO-3100, VisualSonics) was placed over a shaved hindlimb with the animal lying with a 0% tilt to avoid gravity induced fluid shifts.³⁰ Acquisitions were performed before, and on days 2, 5, 7, 13, and 15 following BTX injections. Animals were anesthetized (3% isoflurane, 2 L/min O₂), warmed with an infrared lamp and the heart rate was maintained at 500-550 beats/min. For vascular imaging for injected (left) and contralateral (right) limbs the transducer was placed above and parallel to the femoral, popliteal, and saphenous arteries. The collected measurements were: velocity time integral (VTI, mm/stroke), a measure of blood velocity equivalent to the area under the velocity time curve of each pulse; mean velocity (mm/s), mean gradient (mmHg), peak velocity (PV) (mm/s), and peak gradient (mmHg). The variables from the same sonography curves were highly correlated (>0.90), therefore only VTI is shown. The diameter of the femoral artery (mm) was measured, allowing to quantify stroke volume (ml) and blood flow (ml/min) in the femoral artery. Echocardiography was done before and on days 9 and 19 after BTX injection (Figure 1A) as described previously.³¹

Microfil contrast agent

On day 21, animals were euthanized and immediately perfused using a peristaltic pump with 50 mL of warm (37 °C) heparinized saline (100 U heparin/mL) through the left ventricle to maintain vessel integrity and prevent blood clotting, followed by 10% neutrally buffered formalin for fixation. Then, a 10 mL mixture of MicroFil yellow contrast agent (Flow Tek Inc.) and curing agent was prepared and introduced to the vasculature as previously described.³² Following perfusion, cadavers were placed at 4 °C overnight to allow for complete curing of contrast agent. Whole hindlimbs were dissected, stored in 70% ethanol, and imaged using ex-vivo microCT (Skyscan 1272, 70 kVp, 138 μA, 0.4° rotation step, 0.5 mm Al filter) to collect 5 μm isometric voxel resolution images of the entire hindlimb using batch scanning. Reconstructed images were imported into XamFlow software (Lucid Concepts AG) and the volume of Microfil-filled vessels was quantified by first using Otsu segmentation to select all bone and perfused vasculature as they have similar densities. Then, the largest object (ie, tibia and fibula) was removed, and any remaining regions of bone were segmented out. The total hindlimb volume was calculated by segmenting the soft tissue and bone from the background and filling any small gaps in the selected region. Analysis was done for the volume of the filled vasculature along the length of the tibia, normalization to total hindlimb volume accounted for reduced soft tissue volume in BTX-injected limbs.³³

Synchrotron radiation imaging of osteocyte lacunar and vascular porosity

Dissected hindlimbs were sealed in 70% ethanol vapor in 5 mL Eppendorf tubes and stabilized using a dense foam containing excess ethanol. Samples were scanned at BM05 at the European Synchrotron Radiation Facility. Images were acquired using an energy of 82 keV with a sample-detector propagation distance of 1.4 μm, to image two volumes of interest, corresponding to those analyzed using microCT. Images were reconstructed using Tomwer (ESRF), with a phase retrieval delta beta ratio of 400 and a Munch ring removal algorithm, with resulting images of 1.4 μm isometric voxel size. Samples were processed using a XamFlow workflow as previously described.³⁴ The bone was divided into the anterior, medial, lateral, and posterior quadrants, based on strain patterns during habitual loading that occur due to the large curvature of murine tibiae.³⁵ The following lacunar properties were measured number density, angle, volume and surface area, and lacunar stretch, which was assessed by fitting an ellipsoid with eigenvalues l₁, l₂, l₃ to each lacunae, and calculating lacunar stretch as the difference between the largest and smallest eigenvalues relative to the largest value ((l₁ − l₃)/l₁).³⁶ The following vascular canal properties were measured: volume, surface area, number, separation, and porosity (vascular volume/bone volume). In addition, we measured total larger porosity, and validated basic microCT measurements.

Statistical analysis

Statistical analysis was performed using SAS software (version 9.4, SAS Institute Inc.). The effect of BTX injection (BTX, Vehicle), on limbs (Injected, Contralateral) and the interaction between these terms were assessed with 2-way ANOVA for values with 2 time points. Ultrasound data was assessed using a 3-way mixed ANOVA, with 2 within-subject factors (time and limb) and 1 between-subjects factor (treatment). Tukey–Kramer post hoc tests were performed for comparisons among groups. Paired t-tests were used to compare injected and contralateral limbs within BTX-injected mice. A p-value of <.05 was considered significant. Data are presented as mean ± SD in tables and graphs. When data are presented as percent changes, they were calculated as (time point − baseline)/baseline^*100.

Results

BTX induced local paralysis and reduced rearing in the mice

To assess and quantify the degree of BTX-induced paralysis, we combined digit abduction and wire hang test scores (Figure 1B). All vehicle-injected mice scored 0 or normal on both digit abduction and wire hang, while the BTX-injected group scored between 3 and 7 on paralysis tests (Figure 1B), indicating that peak paralysis occurred between days 2 and 8 post-injection. After this time, affected animals slowly regained function. By day 21 only one BTX-injected animal had recovered fully (score 0) with others scoring an average of 2.7 (Figure 1B). For further analysis, we averaged the measurements obtained during the peak paralysis (day 2-8) and recovery (day >8) periods. We used a 3D-mounted camera to assess animal activity (Figure 1C, Table S1). No differences in the total distance traveled (Figure 1C, left), activity duration (Figure 1C, middle) or mean speed (not shown) were found between the BTX-and vehicle-injected animals at baseline, peak paralysis, or recovery. Interestingly, rearing activity was significantly reduced in BTX-injected animals (Figure 1C, right) at peak paralysis and during the recovery period. After 21 d, BTX-injected animals lost an average of 11.7 ± 3.3% of their body weight, while vehicle-injected animals gained 1.7 ± 3.9%.

BTX induced extensive tibial bone loss in hindlimbs

To examine the effect of BTX-induced paralysis on tibial bone structure, injected, and contralateral tibias in BTX- and vehicle-injected animals were scanned using in vivo microCT (8 μm voxel size). Changes between baseline (3 d pre-injection) and end of experiment (21 d post-injection) were analyzed. At the mid-diaphysis in the BTX group, the injected and contralateral limbs exhibited a loss of 15% and 9% in cortical area to tissue area, respectively, and 17% and 8% cortical bone thickness, respectively (Figure 2A, Table S2). Consistent with these losses, marrow area increased 23% in injected and 15% in contralateral limb in the BTX-injected group (Figure 2A). As expected, injected and contralateral limbs of the vehicle group did not differ from baseline (Figure 2A). At the cortical bone of the proximal metaphysis region of BTX-injected and contralateral limbs, Ct.Ar/Tt.Ar was reduced by 11% and 9% and Ma.Ar increased by 12% and 8% in injected and contralateral BTX limbs, respectively, while these measures were stable in the vehicle group (Figure 2B, Table S3). An effect of treatment (BTX versus Vehicle; ANOVA term: a) was evident in all cortical parameters assessed at both tibial regions (a, p < .01) by two-way ANOVA, and Tukey–Kramer post-hoc analysis showed significant differences between BTX- and vehicle-injected animals (Figure 2A and B).

Figure 2.

BTX-induced bone loss. (A) Tibial mid-diaphysis, (B) trabecular proximal metaphysis, and (C) cortical proximal metaphysis regions were imaged 3 d before and 21 d after the BTX injection. Cortical (Ct. Ar/Tt.Ar, Ct.Th, ma.Ar, and Ct.Ar) and trabecular parameters (Tb.BV/TV, Tb.Th, Tb.Sp, and Tb.N) were assessed and the changes between timepoints in injected (closed circles) and contralateral (open circles) bones in vehicle-injected (open area) and BTX-injected (shaded area) mice were calculated. Data presented as means ± SD; ANOVA main effects: (a) treatment, (b) limb, and (c) interaction. ^#p < .05; ^##p < .01 indicate significant difference based on Tukey–Kramer post-hoc test.

Trabecular bone in the proximal tibial metaphysis was adversely affected by BTX injection into hindlimb musculature. Trabecular bone volume per tissue volume (Tb.BV/TV) was reduced at 21 d compared to baseline in BTX group in injected and contralateral limbs by 66% and 51%, respectively. Trabecular bone loss in the vehicle group due to physiological aging was 20 ± 6.5% (Figure 2C). Changes in trabecular properties were most evident in the loss of trabecular thickness (Tb.Th), which decreased in BTX group by 9% and 4% in injected and contralateral limbs, respectively, but increased in vehicle group by 10% and 13% in injected and contralateral limbs, respectively. Trabecular separation (Tb.Sp) and trabecular number (Tb.N) did not differ (Figure 2C). Thus, morphological assessment of tibiae in BTX-injected mice demonstrate bone loss in injected and contralateral limbs, causing deterioration at diaphysis and metaphysis regions.

BTX-induced bone loss occurred endocortically

To spatially characterize BTX-induced bone loss, microCT-based timelapse morphometry was performed by registering the reconstructed scans from baseline and day 21 and assessing changes on the periosteal and endocortical surfaces of the tibia. At the mid-diaphysis, bone resorption covered most of the endocortical surface in the BTX-injected limb, while both resorption and formation were evident at the periosteal surface (Figure 3A, Table S4). The tibial endocortical surface of BTX-injected mice demonstrated greater eroding volume. When eroding volume was normalized to bone volume (EV/BV), EV/BV was 10-fold greater in BTX-injected limbs, while eroding surface normalized to bone surface (ES/BS) was 3-fold greater in BTX-injected limb (Figure 3A). In the BTX group contralateral limbs, EV/BV and ES/BS was nearly double that of vehicle contralateral limbs (Figure 3A). Meanwhile, bone formation at the mid-diaphysis was minimal on the endocortical surface in the BTX-injected limb: mineralizing volume fraction (MV/BV) and mineralizing surface fraction (MS/BS) in BTX-injected limbs were 96% less than values observed in vehicle-injected limb, and in the BTX group contralateral limb MV/BV and MS/BS were 80% less than vehicle contralateral limb (Figure 3A).

Figure 3.

3D-registered micro-CT based time-lapse. Morphometry of (A) cortical bone of the tibial mid-diaphysis and (B) trabecular bone of the proximal metaphysis. Left: reconstructions of bone (re)modeling in BTX-injected and vehicle-injected limbs indicating regions where bone was non-changed, quiescent (yellow), formed (blue), and resorbed (red). Right: average eroding volume fraction (EV/BV), eroding bone surface (ES/BS), mineralizing volume fraction (MV/BV), and mineralizing bone surface (MS/BS) for injected (closed circles) and contralateral (open circles) bones in vehicle-injected (open area) and BTX-injected (shaded area) animals on endocortical and periosteal surfaces of cortical bone (A) or on trabecular surfaces (B). Data presented as means ± SD; ANOVA main effects: (a) treatment, (b) limb, and (c) interaction. ^#p < .05; ^##p < .01 indicate significant difference based on Tukey–Kramer post-hoc test. ^*p < .05; ^**p < .01 indicate significant differences between injected and contralateral limbs by paired t-test.

In the proximal metaphysis, cortical bone demonstrated more erosive changes in the BTX group compared to vehicle group (Figure S1). At the endocortical surface, BTX group EV/BV was 5-fold greater than vehicle-injected limb and ES/BS was 1.3-fold higher compared to vehicle-injected limb, while mineralization was suppressed compared to vehicle animals (Figure S1). On the periosteal surface, erosion was greater in BTX group than vehicle, while the mineralization indices were similar (Figure S1). Trabecular bone erosion in the proximal metaphysis, Tb.EV/BV was also greater in the BTX group compared to the vehicle group, while trabecular bone formation was reduced (Figure 3B, Table S5). When assessed by two-way ANOVA, an effect of treatment (BTX vs Vehicle; ANOVA term: a) was evident for all endocortical parameters at both tibial regions, as well as for bone resorption at the periosteal surface of the proximal metaphysis (p < .05), with Tukey–Kramer post-hoc analysis showing significant differences between BTX- and vehicle-injected animals (Figure 3A and B). Taken together, 3D-time-lapse morphometry demonstrates that bone deterioration occurred predominantly at the endocortical surface of the tibia.

Vascular flow alterations after BTX-injection

To assess temporal patterns in the mouse hindlimb blood vascularity, in vivo ultrasound imaging was conducted prior to injection and on days 2, 5, 7, and 13 and 15 following injections. Three main arteries (femoral, saphenous, and popliteal) were imaged at approximately the same location along the vessel’s length in all animals (Figure 4A, Tables S6 and S7). Once located, multiple pulse waves were recorded, and vascular parameters were measured (Figure 4A). Data from various days were combined into 3 periods of paralysis identified in behavioral study (Figure 1B), and labels are representative of baseline (day 0), peak paralysis (day 2, 5, 7), and recovery (day 13 and 15).

Figure 4.

Blood flow of main hindlimb arteries. (A) Location of femoral artery (blue) in animal hindlimb as visualized under ultrasound probe (40 MHz) and the corresponding flow averaged over multiple cycles. (B-D) the velocity time integral (VTI, left) and peak velocity (PV, right) of blood in femoral (B), popliteal (C), and saphenous (D) arteries during baseline (day 0), peak paralysis (day 2-8), and recovery (day >8) for injected (closed circles) and contralateral (open circles) bones in vehicle-injected (open area) and BTX-injected (shaded area) animals. Data presented as means ± SD; ANOVA main effects: (a) treatment, (b) limb, and (c) period. ^#p < .05; ^##p < .01 indicate significant difference based on Tukey–Kramer post-hoc test. ^*p < .05; ^**p < .01 indicates significant difference between injected and contralateral limbs by paired t-test.

The femoral artery is the main conduit artery supplying the hindlimb, and it is sufficiently large for a reliable size measurement in our experimental setup. The diameter of the femoral artery at the bifurcation did not change in any treatment group or limb throughout the study (Table S6 and S7), suggesting that an increase in blood velocity indicates an increase in blood flow in the femoral artery. Femoral stroke volume and femoral cardiac output followed the same trend as the other parameters measured in the femoral artery (Figure S2). At baseline, there were no differences in the VTI in the femoral artery in the limbs of BTX (injected 6.12 ± 0.66 mm, contralateral 6.89 ± 1.62 mm) and vehicle (injected 6.57 ± 1.07 mm, contralateral 7.07 ± 0.83 mm) groups (Figure 4B). During peak paralysis (days 2, 5, and 7 post-injection), BTX-injected mice exhibited increased VTI in the injected limb by 20% from baseline and decreased VTI in the contralateral limb by 29% from baseline with significant differences between the limbs (p < .01). In contrast, the VTI in the femoral artery in vehicle animals decreased in both limbs from baseline by 7% and 18% in injected and contralateral limbs, respectively (Figure 4B). During the recovery period (day 13, 15 post-injection), femoral artery VTI in BTX animals remained significantly lower in the contralateral limb (35% difference from baseline) but normalized in the injected limb (3% difference from baseline). The femoral artery VTIs of limbs of vehicle-injected mice were 16%-25% lower than their baseline values during this recovery period (Figure 4B). Similar alterations over time were noted in other femoral vascular parameters, including PV, which was elevated by 17% in BTX-injected limb compared to a 20% decrease from baseline in contralateral limb at peak paralysis. In vehicle-injected animals, PV at baseline, peak paralysis, and recovery did differ between injected and contralateral (Figure 4B). When assessed by three-way mixed ANOVA (time and limb as within-subject factors and treatment as between-subjects factor), there was a significant effect of the period (ANOVA term: c) on which measurements are taken (c, p < .05). Additionally, interactions were found between the limb (Injected vs Contralateral; ANOVA term: b; and period; ANOVA term: c) (b^*c, p < .01) and between the treatment and limb (a^*b, p < .05) in both VTI and PV datasets, however post-hoc Tukey–Kramer analysis identified significant differences between BTX and vehicle animals for VTI but not PV. Paired t-test analysis demonstrated significant differences between injected and contralateral limbs in the BTX group, but not the vehicle group (Figure 4B). Therefore, within the BTX-injected mouse, blood flow was elevated in the injected limb and reduced in the contralateral limb, while both limbs in vehicle-injected mice exhibited similar flow over time.

The popliteal and saphenous arteries arise from the femoral bifurcation. The popliteal artery is responsible for deep muscle and tibial bone supply, while the saphenous artery supplies superficial medial musculature and skin of the hindlimb.⁷ At baseline, VTI and PV were similar in BTX and vehicle groups in popliteal (Figure 4C) and saphenous (Figure 4D) arteries. During peak paralysis, an increase in VTI, but not in PV, was evident for BTX injected limbs in popliteal artery, while these parameters were similar in saphenous artery. At recovery, the VTI in popliteal and saphenous arteries were affected in BTX-injected animals. Specifically, and similar to the femoral artery, popliteal artery VTI was lower in the contralateral limb than the injected limb of BTX group (Figure 4C). In the saphenous artery, the contralateral limb had a higher VTI than the injected limb (Figure 4D). When assessed by three-way mixed ANOVA, there was a significant effect of period on which measures are taken (c, p < .01) for all parameters. In the popliteal VTI dataset, a significant effect of limb as well as interaction between limb and time were evident (b, b^*c, p < .05). Paired t-test showed significant differences between injected and contralateral limbs in the BTX group at peak paralysis (VTI) and recovery (VTI, PV). The blood velocity in both arteries did not respond similarly to BTX as the femoral artery section, with higher velocities continuing from femoral to popliteal arteries.

We have performed echocardiography prior to injection and at days 9 and 19 after injection. At baseline, there were no differences between groups, except for left ventricular mass (LVmass) normalized to tibia length, which was higher in vehicle group (p < .05) (Table S8). Given a difference at baseline, we have analyzed changes at days 9 and 19 relative to baseline (Figure S3). According to ANOVA, there were no differences in fractional shortening (FS) and relative wall thickness (RWT), while aortic and pulmonary VTI demonstrated significant effect of time. LVmass/TibiaLength was the only parameter that demonstrated significant treatment effect and the interaction between treatment and time (Figure S3). Further studies to confirm alterations in LVmass are needed given baseline differences observed in our cohorts despite the randomization.

Vessel architecture in the hindlimb

MicroFil-perfused vessels between the proximal and distal ends of the tibia were assessed with microCT in BTX- and vehicle-injected limbs (Figure 5A). Twenty-one days after injection, the blood vessel volume in BTX-injected limbs was 31% lower than in the vehicle-injected limbs (p < .05) (Figure 5B). However, the limb’s total volume (including hard and soft tissues) was 43% less in BTX-injected limb than in vehicle-injected limbs (p < .01) (Figure 5C). Normalized vessel volume per the total hindlimb tissue volume was similar between the groups (Figure 5D).

Figure 5.

Contrast agent infused vessels. (A) Reconstructed 3D representation of Microfil-filled vessels in hindlimb of vehicle and BTX-injected limbs. (B) The volume of filled vessels, (C) total tissue volume, and (D) ratio of vessels volume (VV) to tissue volume (TV) in vehicle-injected (white circles) and BTX-injected (black circles) hindlimbs. Data presented as means ± SD; ^*p < .05; ^**p < .01 indicate significant difference between groups by unpaired t-test.

Lacunar and vascular porosity

To visualize bone osteocyte lacunar and vascular porosities in limbs, the tibias of BTX and vehicle-injected limbs were imaged using synchrotron radiation tomography at the mid-diaphysis and proximal metaphysis regions. Lacunar and vascular porosities were analyzed for the full volume of bone and among four quadrants (anterior, posterior, medial, and lateral) at mid-diaphysis (Figure 6A) and at proximal metaphysis (Figure 7A). In the mid-diaphysis, lacunar properties of the whole volume and four quadrants did not differ between BTX- and vehicle-injected limbs (Figure 6B). In contrast, vascular surface area to bone volume (VSa/BV) and vascular volume to bone volume (VV/BV) were lower in BTX-injected limbs by 22% and 24%, respectively, compared to vehicle-injected limbs (p < .05) (Figure 6C). This difference was most pronounced in the posterior quadrant of the mid-diaphysis, which displayed 50% lower vascular porosity in BTX-injected mice compared to vehicle-injected limb posterior quadrant (Figure 6C).

Figure 6.

Osteocyte lacunar and vascular network in tibia mid-diaphysis. (A) Cortical bone segmentation into 4 quadrants (anterior, posterior, lateral, and medial) from the center of mass of each sample (left) and 3-D reconstruction of osteocytes (green) and vascular pores (red) in vehicle-injected and BTX-injected bone (right). (B) Lacunar properties (volume, stretch, density) between vehicle-injected (open circle) and BTX-injected (closed circle) mice in full volume and 4 color-coded quadrants. (C) Vascular pore properties (surface area to bone area, volume to bone area, thickness) in vehicle-injected (open circle) and BTX-injected (closed circle) mice in full volume and 4 color-coded quadrants. Data presented as means ± SD; ANOVA main effects: (a) treatment, (b) quadrant, and (c) interaction. ^#p < .05; ^##p < .01 indicate significant difference based on Tukey–Kramer post-hoc test.

Figure 7.

Osteocyte lacunar and vascular network in tibia proximal metaphysis. (A) Cortical bone segmentation into four quadrants (anterior, posterior, lateral, and medial) from the center of mass of each sample (left) and 3-D reconstruction of osteocytes (green) and vascular pores (red) in vehicle-injected and BTX-injected bone (right). (B) Lacunar properties (volume, stretch, density) between vehicle-injected (open circle) and BTX-injected (closed circle) mice in full volume and 4 color-coded quadrants. (C) Vascular pore properties (surface area to bone area, volume to bone area, and thickness) in vehicle-injected (open circle) and BTX-injected (closed circle) mice in full volume and 4 color-coded quadrants. Data presented as means ± SD; ANOVA main effects: (a) treatment, (b) quadrant, and (c) interaction. ^#p < .05; ^##p < .01 indicate significant difference based on Tukey–Kramer post-hoc test.

In the proximal metaphysis, whole lacunar stretch was higher in the BTX group compared to vehicle group (p < .05) while lacunar density and volume did not differ (Figure 7B). Within the quadrants, the anterior quadrant lower lacunae number density and their stretch was higher indicating flatter and longer lacunae (Figure 7B). Vascular porosity parameters, specifically VV/BV and VSa/BV, were lower by 21% and 17%, respectively, in BTX- compared to vehicle-injected mice (Figure 7C). Similarly, quadrant specific changed were observed in the anterior quadrant of the proximal metaphysis, with an increase in vascular surface area and volume, when indexed to bone volume in this region (Figure 7C).

Discussion

Our study aimed to understand vascular and bone adaptations in the BTX model of mechanical unloading. We observed distinct vascular and bone changes in the BTX-injected and contralateral limbs of skeletally mature (26-wk-old) female mice. In the BTX-injected limb, we identified a significant negative impact on tibial bone structure 3-wk post-injection. Simultaneously, the BTX-injection was associated with an increase in blood flow and velocity in the femoral artery and to a smaller degree increase velocity in the popliteal artery. Despite this increased velocity in the main hindlimb arteries of BTX-injected limbs, bone vascular porosity was significantly diminished suggesting deterioration of bone microvasculature. We noted that BTX-induced surface (endocortical > periosteal) and site (metaphysis > diaphysis) dependant changes in bone morphology as well as quadrant (eg, anterior, posterior, medial, and lateral) dependant changes in osteocyte network of bone. In BTX-injected mice, the contralateral, non-injected limb also demonstrated changes in bone and blood flow properties, including a temporal decrease of blood flow and velocity in the main hindlimb arteries and concurrent bone loss over the 3-wk experiment. Examining the physiologic mechanical loading environment in both groups, we noted similar running speed and distance for BTX-injected and control mice, which may suggest higher mechanical loads on the contralateral limb during ambulation due to paralysis of the injected limb. However, only modest decreases (20%-30%) in ground reaction force were previous reported in BTX-paralyzed limbs.³³ While we did not measure ground reaction forces in this study, we observed sustained impairment in behavioral rearing in BTX-injected mice, likely reducing the frequency of vertical ground forces in both limbs. In summary, BTX-induced paralysis resulted in skeletal and vascular changes in the injected limb due to neuromuscular blockade and altered activity patterns, while changes in the contralateral limb changes may be due to altered activity patterns alone. Future studies are needed to examine the effect of impaired rearing behavior on vascular and bone adaptation in mice.

The observed bone degradation was induced by BTX-mediated blockage of the release of neurotransmitters from peripheral nerve endings in muscle and the subsequent paralysis.³⁷ A recent scoping review of bone loss in BTX-treated animal models suggests a comparable trabecular BV/TV reduction (46%-80%) in studies with similar experimental design as this one.³⁸ Physiological bone loss due to aging observed in our study and reported previously³⁹ is 40% lower than the bone loss in the BTX injected limbs and 20% lower than the bone loss the contralateral limbs of BTX-injected animals in our study. Several studies directly compared BTX-induced bone loss to other models of mechanical unloading. BTX-induced bone loss was found to be higher than in the hindlimb suspension model, and combining both methods resulted in a more deleterious effect.²⁶ Comparison of different models of paralysis suggests higher bone loss following BTX injection than peripheral nerve injury group,³³ but similar bone deterioration following complete sciatic neurectomies as in BTX model.¹² Thus, compared to ambulatory impairment only, disrupting neuronal signaling appear to have an additional negative effect on bone structure, which corresponds to the degree of impairment.

Bone loss in the contralateral limb was described in other BTX studies carried out with similar conditions, although the cause was not elucidated.^26,40 We suggest that this effect could be due to the reduced rearing ability of BTX-injected animals compared to vehicle controls.^26,40 Rearing, the frequent up-righting of rodents in their cage, likely accounts for higher loads on the hindlimb as most of the body mass is being supported by 2 hindlimbs as opposed to 4. Though this activity was not previously examined in BTX studies, differences between injected and contralateral limb ground reaction forces have been assessed.^33,41 These studies demonstrated sustained reduction of ground reaction forces for BTX-injected limb, but either no change³³ or a relatively small transient increase⁴¹ in the ground reaction forces for the contralateral limb. The lack of rearing behavior could also affect the orthostatic response in these animals and subsequently blood flow. In physiological conditions, up-righting results in blood redistribution to the lower part of the body followed by active adaptation through vasoconstriction.⁴² After spaceflight, multiple studies have shown a decreased vasoconstrictor responsiveness in hindlimb vessels.⁴³ Thus, changes in biomechanical environment and blood flow associated with rearing may potentially underlie the observed bone loss in the contralateral limbs of BTX-injected animals. An alternative explanation of bone loss in contralateral limb could be due to weight loss observed in BTX-injected animals. Previous studies demonstrated that weight loss is consistently associated with bone loss, however in these studies bone loss was reported after much higher weight loss (20%-40% compared to 11.7% in our study) and longer follow up (10-12 wk, compared to 3 wk in our study).⁴⁴ The effect of rearing alone needs to be further investigated for its role in bone and vascular maintenance.

We report an increase in femoral artery blood velocity in BTX-injected limbs, which, since the diameter of the femoral artery remaining unchanged at the root, can be translated into an increase in the femoral artery blood flow. Smaller changes of blood velocity were observed in the popliteal artery, but not in the saphenous artery. Unfortunately, methods used in our study did not allow measuring the diameter of smaller downstream, therefore velocity cannot be used to indicate increased flow in these smaller arteries. A BTX-induced increase in blood flow and velocity is consistent with previous studies demonstrating that BTX prevents the release of norepinephrine from presynaptic nerve endings in vascular smooth muscle thereby leading to dose-dependent vasodilation.⁴⁵ The differences in degree of changes in three arteries may be explained by localized action of BTX. Based on previous experimental and clinical use, the toxin’s spread is dose-dependant with 15-30 mm diffusion with 1 U injections.⁴⁶ Given that our site of injection, the quadriceps muscle, is directly adjacent to the femoral artery while the bifurcation of this artery is at the distal end of the muscle,⁷ this likely explains the higher effect on flow in the femoral artery. Since vasculature flow is an important component of bone homeostasis and a prerequisite for new bone formation,⁴⁷ it would be expected that the vasculature would adapt to diminished mechanical loading. Several studies found that the decrease in blood flow following hindlimb suspension is due to attenuated endothelial-dependant vasodilation in the femoral artery.⁴⁸ Similarly, after sciatic nerve injury, blood flow in the femur is reduced at 2 wk.⁴⁹ In conditions of microgravity (ie, spaceflight) associated with reduced hindlimb bone mass, a cephalic fluid shift exists resulting in less blood flow to the hindlimbs.⁵⁰ In contrast to these models, but consistent with an increased blood flow reported in a surgical model of disuse,¹⁶ BTX-injection increased blood flow in the femoral artery, but did not mitigate BTX-induced tissue or bone loss.

Although blood flow was increased in the femoral artery, we observed reduced bone vascular porosity in BTX-injected limbs, while the changes in lacuna-canalicular network were minimal. Currently, multiple reports provide conflicting results in adaptation of lacunar and vascular pores in BTX-injected hindlimbs. In long term follow up of BTX-injected rats, femoral lacunar density and volume did not differ between injected, contralateral, and control limbs.²² In another study assessing the proximal metaphysis in rats, lacunar density in the whole cortex was reduced after BTX injection.²³ These differences could be explained by the different regions analyzed across studies, age of the animals and target muscles. To our knowledge, our study is the first to explore these lacunar alterations in a mouse model and no one has previously examined vascular porosity of bone after BTX injection. A potential increase in bone vascular porosity after hindlimb suspension or sciatic nerve resection could further explain the coincident decrease in blood flow for these conditions, in contrast to what we have observed after BTX injection: decreased bone vascular porosity and increased blood flow. However, further studies are needed to characterize vascular porosity after hindlimb suspension or sciatic nerve resection. These data suggest complex changes in blood flow and vascularization following BTX injection, supporting the importance of crosstalk between neuronal signaling and mechanical forces in bone maintenance.

Our study has several limitations. The BTX-injected mice in our studies exhibit a noticeable weight loss. Although the degree of weight loss is consistent with other studies in this model that reported 5%-14% weight loss^26,40 this represents a confounder that will need to be addressed in the future studies. The precision of methods used to measure vessel diameter was limited in our studies. As a consequence, it was not always possible to infer changes in blood flow based on the measured changes in blood velocity. Another limitation was a misalignment of the ultrasound measures of blood dynamics that were performed throughout the study, and bone changes that were only assessed at the endpoint, making it difficult to directly relate blood dynamic and bone turnover changes. The bone endpoint of 3 wk after BTX injection potentially missed maximal bone loss, as BTX-induced resorption occurs within the first 2 wk. Further studies are required to resolve these limitations.

Our study demonstrates that the BTX model presents with unique combination of changes in muscular, bone, and vascular environment of hind limbs. We report macro- and micro-vascular adaptation in BTX-injected limbs. Other experimental models demonstrate bone loss following interventions that include mechanical unloading, including hind limb suspension, peripheral nerve injury or neurectomy, casting, and BTX injection. Comparing the degree and timing of changes in different physiological compartments observed in these models will allow reconstructing the pathological sequelae leading to bone loss, thus providing novel approaches to developing countermeasures for spaceflight crew and patients affected by paralysis.

Author contributions

Mahmoud S. Moussa and Taylor de Vet contributed equally to the manuscript.

Bettina M. Willie and Svetlana V. Komarova contributed equally to the study supervision.

Mahmoud S. Moussa (Investigation, Methodology, Data curation, Formal analysis, Validation, Visualization, Project administration, Writing—original draft, Writing—review & editing), Taylor de Vet (Investigation, Methodology, Data curation, Formal analysis, Validation, Visualization, Project administration, Writing—original draft, Writing—review & editing), Nadine Lebcir (Investigation, Formal analysis, Writing—review & editing), Paul Zaslansky (Methodology, Formal analysis, Resources, Supervision, Writing—review & editing), Lorraine E. Chalifour (Methodology, Formal analysis, Resources, Supervision, Writing—review & editing), Bettina M. Willie (Conceptualization, Formal analysis, Funding acquisition, Methodology, Resources, Project administration, Supervision, Writing—review & editing), and Svetlana V. Komarova (Conceptualization, Formal analysis, Funding acquisition, Methodology, Resources, Project administration, Supervision, Writing—review & editing).

Funding

This study was funded by grants from Canadian Space Agency #21HLSRM03, Natural Sciences and Engineering Research Council #RGPIN-28825, and Canadian Institute for Health Research PJT-183656..

Conflicts of interest

None of the authors have any perceived or actual conflicts of interest to report.

Data availability

The aggregated data underlying this article are available in the article and in its online supplementary material. The individual animal data will be shared on reasonable request to the corresponding author.

Statement relating to ethics and integrity policies

All animal use in this project was approved by the McGill Animal Compliance Office (ACO) as well as the Facility Animal Care Committees at the Shriner’s Facility and Jewish General Hospital (JGH) (AUP#2020-8192).