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

Figures

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  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.

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  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.

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  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.

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  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.

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  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.

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  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.

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  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.