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.

Keywords: blood flow; bone loss; botulinum toxin A; mechanical unloading; osteocyte network analysis; synchrotron imaging; vascular porosity.

Graphical Abstract

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.

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.

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.

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.

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.

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.