Eccentric Contraction-Induced Muscle Injury: Reproducible, Quantitative, Physiological Models to Impair Skeletal Muscle's Capacity to Generate Force

Abstract

In order to investigate the molecular and cellular mechanisms of muscle regeneration an experimental injury model is required. Advantages of eccentric contraction-induced injury are that it is a controllable, reproducible, and physiologically relevant model to cause muscle injury, with injury being defined as a loss of force generating capacity. While eccentric contractions can be incorporated into conscious animal study designs such as downhill treadmill running, electrophysiological approaches to elicit eccentric contractions and examine muscle contractility, for example before and after the injurious eccentric contractions, allows researchers to circumvent common issues in determining muscle function in a conscious animal (e.g., unwillingness to participate). Herein, we describe in vitro and in vivo methods that are reliable, repeatable, and truly maximal because the muscle contractions are evoked in a controlled, quantifiable manner independent of subject motivation. Both methods can be used to initiate eccentric contraction-induced injury and are suitable for monitoring functional muscle regeneration hours to days to weeks post-injury.

Keywords: Force drop; Lengthening contraction; Muscle damage.

Fig. 1

(a) In vitro muscle physiology system and (b) custom, 1.2 ml organ bath with electrodes sized for mouse soleus or EDL muscle. The in vitro system described here is similar to that described by Sperringer and Grange in Chapter 19 of this volume [8], particularly in regard to the lever system

Fig. 2

Dissection of mouse EDL muscle. Orientation is such that in (a)–(d) the foot of the mouse is at the top of the picture. (a) Distal tendon of the tibialis anterior (TA) muscle is separated from the distal EDL tendon by forceps. (b) Distal TA tendon is cut exposing EDL muscle. Blade of Vannas scissors is shown cutting proximal TA muscle to expose medial EDL tendon. Blue line demarks midline of the EDL muscle. PT = patellar tendon. (c) Suture placed under distal EDL tendon, precisely at myotendinous junction. Note that tendon has been cleared of connective tissue and fat. (d) Both distal tendon (top) and medial tendon (bottom) of the EDL muscle are secured by suture. We prefer to tie sutures on the muscle in vivo to keep muscle at its anatomical length as long as possible during the dissection and to minimize handling of the muscle ex vivo. (e) Once completely excised, gel type cyanoacrylate adhesive is carefully applied with a needle (arrow) to cover tendons and knots of the suture to avoid slippage during eccentric contractions

Fig. 3

(a) Representative length-time and (b) force-time tracings of a single in vitro eccentric contraction by a mouse EDL muscle. (c) A series of five eccentric contractions can be titered by using only 10 % L_o length change at a slow velocity of 0.75 L_o/s to result in no force loss in healthy muscle (wt) but substantial force drop in muscle lacking dystrophin (mdx) and muscle lacking both dystrophin and utrophin (dko). This establishes an optimal situation to detect therapeutic improvement in the disease models. (d) In vitro contraction-induced injury shown as a loss in maximal isometric tetanic force (Po) for mdx mouse muscle following eccentric contractions (Post) as compared to before those injurious contractions (Pre). Note, though the force is typically measured in grams (g), it should be converted to newton (N), which is the SI unit of force for reporting. For example, an 8 mg EDL mouse muscle should generate ~35 g, i.e., 343 mN, of force. Notice that the peak-eccentric force (in b) is 150–175 % of peak-isometric force (Pre in d)

Fig. 4

(a) The custom in vivo platform with dimensions. (b) Optimal placement of the mouse on the platform. The mouse position is secured with adhesive tape, and the self-adhesive temperature probe lies underneath the animal. (c) Appropriate electrode placement on either side of the peroneal nerve for dorsiflexion. (d) Appropriate electrode placement on either side of the sciatic nerve for plantarflexion

Fig. 5

Representative torque-time tracing of a single in vivo eccentric contraction performed by the plantar-flexors. The length change reflects the foot being moved from 20° plantarflexion to 20° dorsiflexion during the electrical stimulus for contraction. The torque tracing demonstrates an ~100 % increase in torque production during the eccentric portion of the contraction compared to the isometric portion

Fig. 6

In vivo contraction-induced injury by the dorsi-flexors (a) and plantar-flexors (b) among C57Bl controls (wt), dystrophin deficient (mdx), dystrophin deficient and utrophin heterozygous (het), and dystrophin deficient and utrophin overexpression (Fiona) mice [15]. (c) In vivo contraction-induced injury controlled to a 25 % loss of torque in wt and mdx muscles. (d) Lack of recovery of dorsiflexion and plantarflexion torque during the hour immediate post-injury demonstrates injury as opposed to fatigue. (e) Longitudinal recovery of dorsiflexion torque as a function of stimulation frequency after a severe eccentric contraction-induced injury. (f) Longitudinal recovery of dorsiflexion torque as a function of stimulation frequency after a relatively mild eccentric contraction-induced injury