Long-Term Quantitative Evaluation of Muscle and Bone Wasting Induced by Botulinum Toxin in Mice Using Microcomputed Tomography
Muscle and bone masses are highly correlated and muscles impose large loads on bone. Muscle wasting that accompanies bone loss has been poorly investigated. 21 female mice were spread into seven groups. At day 0, 18 mice received Botulinum toxin (BTX) injection in the quadriceps muscle to induce paralysis of the right hind limb; the left contralateral side was used as control. Mice were sacrificed at 7, 14, 21, 28, 56 and 90 days post-injection. A remaining group was sacrificed at day 0. Trabecular bone volume was determined by microcomputed tomography (microCT) at the distal femur and tibia proximal metaphyses on both sides. Limbs were immersed in an HgCl2 solution allowing muscle visualization by microCT. On 2D sections, the cross-sectional areas and form-factors were measured for the quadriceps at mid-thigh and gastrocnemius at mid-leg and these muscles were dissected and weighed. Bone volume decreased in the paralysed side. Bone loss was maximal at 56 days followed by recuperation at 90 days. The cross-sectional areas of gastrocnemius and quadriceps were significantly lower in the paralysed limb from 7 days; the decrease was maximum at 21 days for the gastrocnemius and 28 days for the quadriceps. No difference in form-factors was found between the two limbs. Similar results were obtained with the anatomical method and significant correlations were obtained between the two methods. Quantitative analysis of muscle loss and recovery was possible by microCT after using a metallic contrast agent. Loss of bone secondary to muscle wastage induced by BTX and recovery showed a parallel evolution for bone and muscles.
KeywordsBotulinum toxin Bone loss MicroCT Muscle loss Heavy metal staining
This work was made possible by grants from the French Ministry of Research. Authors thank P. Legras and J. Roux for their help with the animal care (SCAHU) and L. Techer for secretarial assistance. Mrs. N. Gaborit and S. Lemière are thanked for their technical help with microCT analysis. Mr. G. Mabilleau is thanked for reviewing the English manuscript.
HL performed animal experiments and legs handling, wrote parts of the manuscript; CG and EA performed anatomical dissections of the mice and muscle weighing. NM performed the morphometric analysis of muscles on VG Studiomax; DC designed the study, analysed all microCT data, did all statistical analyses and wrote the manuscript. All authors have read and approved the final manuscript.
Compliance with Ethical Standards
Conflict of interest
Hélène Libouban, Claude Guintard, Nicolas Minier, Eric Aguado and Daniel Chappard declare that they have no conflict of interest.
Human and Animal Rights and Informed Consent
Animal care and experimental protocols were approved by the French Ministry of Research and were done in accordance with the institutional guidelines of the French Ethical Committee (protocol agreement number 01732.01) and under the supervision of authorized investigators.
Video Clip 1: (video_clip2_right_side.wmv) MicroCT analysis of the muscle anatomy of a full leg after contrast enhancement in HgCl2. 3D reconstruction image of the left (control) side of a mouse at 21 days. The 3D images were produced with a volume rendering software (CTVox—Skyscan) with a look-up table emphasising muscle in red and bone in white (MP4 3874 KB)
Video Clip 2: (video_clip2_right_side.wmv) MicroCT analysis of the muscle anatomy of a full leg after contrast enhancement in HgCl2. 3D reconstruction image of the right side of the same animal which received a BTX injection in the right Musculus quadriceps femoris. The 3D images were produced with a volume rendering software (CTVox—Skyscan) with a look-up table emphasising muscle in red and bone in white (MP4 3780 KB)
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