Restriction of Postoperative Joint Loading in a Murine Model of Anterior Cruciate Ligament Reconstruction: Botulinum Toxin Paralysis and External Fixation

Abstract

Control of knee motion in small animal models is necessary to study the effect of mechanical load on the healing process. This can be especially challenging in mice, which are being increasingly used for various orthopedic reconstruction models. We explored the feasibility of botulinum toxin (Botox; Allergan, Dublin, Ireland) paralysis and a newly designed external fixator to restrict motion of the knee in mice undergoing anterior cruciate ligament (ACL) reconstruction. Nineteen C57BL/6 mice were allocated to two groups: (1) Botox group (n = 9) and (2) external fixator group (n = 10). Mice in Botox group received two different doses of Botox: 0.25 unit (n = 3) and 0.5 unit (n = 6). Injection was performed 72 hours prior to ACL reconstruction into the quadriceps, hamstring, and calf muscles of the right hind leg. Mice in external fixator group received an external fixator following ACL reconstruction. Mice were monitored for survival, tolerance, and achievement of complete knee immobilization. All mice were meant for sacrifice on day 14 postoperatively. No perceptible change in gait was observed with 0.25 unit of Botox. All mice that received 0.5 unit of Botox had complete hind limb paralysis documented by footprint analysis 2 days after injection but failed to tolerate anesthesia and were euthanized 24 hours after operation due to their critical condition. In contrast, the external fixator was well tolerated and effectively immobilized the limb. There was a single occurrence of intraoperative technical error in the external fixator group that led to euthanasia. No mechanical failure or complication was observed. Botox paralysis was not a viable option for postoperative restriction of motion and joint loading in mice. However, external fixation was an effective method for complete knee immobilization and can be used in murine models requiring postoperative control of knee loading. This study introduces a robust research tool to allow control of postoperative joint loading in animal models such as ACL reconstruction, permitting study of the effects of mechanical load on the biologic aspects of tendon-to-bone healing.

Keywords

murine - anterior cruciate ligament - immobilization - external fixation - Botox

• References

• 1 Józsa L, Réffy A, Järvinen M, Kannus P, Lehto M, Kvist M. Cortical and trabecular osteopenia after immobilization. A quantitative histological study of the rat knee. Int Orthop 1988; 12 (02) 169-172
• 2 Jee WS, Ma Y. Animal models of immobilization osteopenia. Morphologie 1999; 83 (261) 25-34
• 3 Kaspar K, Schell H, Toben D, Matziolis G, Bail HJ. An easily reproducible and biomechanically standardized model to investigate bone healing in rats, using external fixation. Biomed Tech (Berl) 2007; 52 (06) 383-390
• 4 Mark H, Bergholm J, Nilsson A, Rydevik B, Strömberg L. An external fixation method and device to study fracture healing in rats. Acta Orthop Scand 2003; 74 (04) 476-482
• 5 Sigurdsen UE, Reikeras O, Utvag SE. External fixation compared to intramedullary nailing of tibial fractures in the rat. Acta Orthop 2009; 80 (03) 375-379
• 6 Stasiak ME, Wiznia D, Alzoobaee S. , et al. A novel device to apply controlled flexion and extension to the rat knee following anterior cruciate ligament reconstruction. J Biomech Eng 2012; 134 (04) 041008
• 7 Hettrich CM, Rodeo SA, Hannafin JA, Ehteshami J, Shubin Stein BE. The effect of muscle paralysis using Botox on the healing of tendon to bone in a rat model. J Shoulder Elbow Surg 2011; 20 (05) 688-697
• 8 Langenskiöld A, Michelsson JE, Videman T. Osteoarthritis of the knee in the rabbit produced by immobilization. Attempts to achieve a reproducible model for studies on pathogenesis and therapy. Acta Orthop Scand 1979; 50 (01) 1-14
• 9 Batt JA, Bain JR. Tibial nerve transection - a standardized model for denervation-induced skeletal muscle atrophy in mice. J Vis Exp 2013; (81) e50657
• 10 Grimston SK, Silva MJ, Civitelli R. Bone loss after temporarily induced muscle paralysis by Botox is not fully recovered after 12 weeks. Ann N Y Acad Sci 2007; 1116: 444-460
• 11 Warner SE, Sanford DA, Becker BA, Bain SD, Srinivasan S, Gross TS. Botox induced muscle paralysis rapidly degrades bone. Bone 2006; 38 (02) 257-264
• 12 Kaplan SJ, Hayes WC, Mudan P, Lelli JL, White AA. Monitoring the healing of a tibial osteotomy in the rabbit treated with external fixation. J Orthop Res 1985; 3 (03) 325-330
• 13 Terjesen T. Bone healing after metal plate fixation and external fixation of the osteotomized rabbit tibia. Acta Orthop Scand 1984; 55 (01) 69-77
• 14 Terjesen T. Healing of rabbit tibial fractures using external fixation. Effects of removal of the fixation device. Acta Orthop Scand 1984; 55 (02) 192-196
• 15 Morse A, Yu NY, Peacock L. , et al. Endochondral fracture healing with external fixation in the Sost knockout mouse results in earlier fibrocartilage callus removal and increased bone volume fraction and strength. Bone 2015; 71: 155-163
• 16 Brophy RH, Kovacevic D, Imhauser CW. , et al. Effect of short-duration low-magnitude cyclic loading versus immobilization on tendon-bone healing after ACL reconstruction in a rat model. J Bone Joint Surg Am 2011; 93 (04) 381-393
• 17 Bedi A, Kovacevic D, Fox AJ. , et al. Effect of early and delayed mechanical loading on tendon-to-bone healing after anterior cruciate ligament reconstruction. J Bone Joint Surg Am 2010; 92 (14) 2387-2401
• 18 Stone AV, Ma J, Whitlock PW. , et al. Effects of Botox and Neuronox on muscle force generation in mice. J Orthop Res 2007; 25 (12) 1658-1664
• 19 Gilday SD, Casstevens EC, Kenter K, Shearn JT, Butler DL. Murine patellar tendon biomechanical properties and regional strain patterns during natural tendon-to-bone healing after acute injury. J Biomech 2014; 47 (09) 2035-2042
• 20 Boivin GP, Elenes EY, Schultze AK, Chodavarapu H, Hunter SA, Elased KM. Biomechanical properties and histology of db/db diabetic mouse Achilles tendon. Muscles Ligaments Tendons J 2014; 4 (03) 280-284
• 21 Hettrich CM, Gasinu S, Beamer BS. , et al. The effect of mechanical load on tendon-to-bone healing in a rat model. Am J Sports Med 2014; 42 (05) 1233-1241
• 22 Wall ME, Banes AJ. Early responses to mechanical load in tendon: role for calcium signaling, gap junctions and intercellular communication. J Musculoskelet Neuronal Interact 2005; 5 (01) 70-84
• 23 Vogel KG. Tendon structure and response to changing mechanical load. J Musculoskelet Neuronal Interact 2003; 3 (04) 323-325 , discussion 333–334
• 24 Banes AJ, Horesovsky G, Larson C. , et al. Mechanical load stimulates expression of novel genes in vivo and in vitro in avian flexor tendon cells. Osteoarthritis Cartilage 1999; 7 (01) 141-153
• 25 Wray Jr RC, Ollinger H, Lowrey R, Weeks PM. Effect of continuous load on the mechanical properties of tendon adhesions. Hand 1981; 13 (01) 92-96