Applications of <em>In Vivo</em> Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Abstract

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

“…In the test presented, the tetanic curve starts at ~60 Hz, where the potentiation can be visualized (Figure 4A) and the maximum force is determined at ~150 Hz (Figure 4B), when the plateau is reached with a completed fused curve 9,16 . Any variation of these results may indicate that the muscles are not being properly stimulated by the electrodes.…”

“…Skeletal muscle function, especially in small rodents, is typically performed using three well-described procedures 8,9 to detect impaired force production and/or monitor disease progression. (1) Ex vivo; where muscle is removed from the animal and incubated in a Ringer's bath solution to assess the muscle function using field stimulation 10 .…”

“…(2) In situ; where the proximal attachment of the muscle remains in the animal and the distal tendon is connected to a force transducer, allowing muscle function to be performed by direct nerve stimulation 11 . (3) In vivo; where electrodes are placed subcutaneously to obtain nerve-evoked muscle force production 9,12 . While these three procedures are used for different purposes, they each possess advantages and disadvantages.…”

Analysis and Modeling of Rat Gait Biomechanical Deficits in Response to Volumetric Muscle Loss Injury

“…In vivo force testing was performed as previously described (Mintz et al, 2016). Briefly, at −1, 2, 4, and 8 weeks relative to the surgery date, rats were anesthetized and the right hind limb was aseptically prepared.…”

“…The biological relevance of the TA VML injury has been previously shown (Sicari et al, 2012; Wu et al, 2012; Aurora et al, 2014) and the rat model of VML injury is advantageous because methods have been established for assessment of force generation ability (Wu et al, 2012; Aurora et al, 2014; Corona et al, 2015; Mintz et al, 2016). In addition, rats have been successfully utilized as models to predict physiological changes in humans for multiple pathologies including cardiovascular disease (Iannaccone and Jacob, 2009), osteoarthritis (Clarke et al, 1997; Ängeby Möller et al, 2012), spinal cord injury (Canu and Garnier, 2009; Perrot et al, 2009; Johnson et al, 2012; Datto et al, 2016), and Parkinson's (Deumens et al, 2002; Duty and Jenner, 2011).…”