The sternomastoid (SM) muscle plays an important role in supporting breathing. It also has unique anatomical advantages that allow its wide use in head and neck tissue reconstruction and muscle reinnervation. However, little is known about its contractile properties. The experiments were run on rats and designed to determine in vivo the relationship between muscle force (active muscle contraction to electrical stimulation) with passive tension (passive force changing muscle length) and two parameters (intensity and frequency) of electrical stimulation. The threshold current for initiating noticeable muscle contraction was 0.03 mA. Maximal muscle force (0.94 N) was produced by using moderate muscle length/tension (28 mm/0.08 N), 0.2 mA stimulation current, and 150 Hz stimulation frequency. These data are important not only to better understand the contractile properties of the rat SM muscle, but also to provide normative values which are critical to reliably assess the extent of functional recovery following muscle reinnervation.
The sternocleidomastoid (SCM) muscle lies on the lateral side of the neck. Anatomically, it is composed of two bellies, a medially and superficially localized sternomastoid (SM), and a laterally and deeply positioned cleidomastoid (CM). Functionally, the SCM participates in head movements and respiration [
As the SM belly is located more superficially in the neck and has a relatively larger muscle mass when compared with the CM belly, it has been widely used as a muscle or myocutaneous flap for reconstruction of oral cavity and facial defects [
We have a longstanding interest in the development of novel surgical techniques to effectively reinnervate paralyzed muscles as the presently used reinnervation methods result in poor outcomes (for review see [
A number of morphological and physiological approaches have been used to assess the success of axonal regeneration and the extent of functional recovery of a reinnervated muscle after a given reinnervation procedure. Electromyography (EMG) [
Although some researchers investigated in vivo SM muscle force in rabbits [
The present study is focused on determining the muscle force characteristics of the SM muscle in healthy rats. These results would provide normative data which could be useful for understanding the physiological role of the SM muscle and for evaluating the extent of functional recovery after reinnervation of a paralyzed SM muscle. We would also like to establish the optimal stimulation parameters which could be used to produce the strongest isometric force by this muscle.
Twelve adult (3.5 months old) Sprague-Dawley male rats (Charles River Laboratories, MA), weighing 350–450 grams, were used in this study. Previous studies [
All rats underwent open neck surgery under an Olympus SZX12 Stereo zoom surgical microscope (Olympus America Inc., Center Valley, PA) to expose the right SM muscle through a midline skin incision from the hyoid bone to the sternum. Animals were anesthetized with an initial intraperitoneal injection of ketamine (80 mg/kg body wt) and xylazine (5 mg/kg body wt); supplementary doses were administered as needed to maintain an adequate state of anesthesia. The rat was placed on a heating pad (homeothermic blanket system, Stoelting, Wood Dale, IL) to maintain its temperature at
Our studies have demonstrated that the rat SM is supplied by a branch derived from the spinal accessory nerve and has a single motor endplate band at the midpoint of the muscle (data not shown). The right SM muscle and its innervating nerve were isolated from surrounding tissues and prepared for nerve stimulation and force measurement. First of all, the rostral tendon of the SM was identified, transected, and attached with a 2-0 suture to a servomotor lever arm (Model 305B Dual-Mode Lever Arm System, Aurora Scientific Inc., Aurora, Ontario, Canada, see Figure
A diagram of the data acquisition system, which provides electrical stimulation and records muscle force. Note that a dell laptop with user written software in Labview 8.2 is used to control the experiment. The SM muscle is detached from its rostral tendon and attached to the lever of servomotor, which controls muscle stretch and measures muscle force. Electrical stimulation with parameters controlled by LabView software (National Instruments) is generated by the Multifunctional board 6251 (National Instruments) and delivered to the SM nerve. Data are analyzed off line with DIAdem 11.0 software (National Instruments).
Isometric contraction of the SM muscle was obtained with two 200 ms trains of biphasic rectangular pulses (0.2 msec duration) separated by a 20-second break. A break of at least 1 minute was used before trying subsequent pairs of trains. The maximum value of muscle force during each 200 ms contraction was identified. The maximal force during the first and second stimulation trains was averaged. Initial passive tension before stimulation was subtracted from this value. This difference between output force and preload force represents the muscle force measurement. Typically, current was set to 0.1 mA and frequency of stimulation to 200 Hz. The force generated by the contraction of the SM muscle was transduced with the servomotor of a 305B lever system and displayed on a computer screen. At the moment of force measurements, the lever arm was stationary.
To prevent cooling and drying, the SM muscle and nerve were regularly bathed with warm mineral oil throughout the testing. Although changes in muscle temperatures above
During force measurement, several parameters influencing force production were examined to establish the optimal settings for obtaining maximum muscle force as described below.
As maximal muscle force can be generated at optimal muscle length, we examined the length-force relationship. Muscle length was controlled by gradually stretching the SM muscle using the lever arm to pull the muscle with different tensions (very low tension—0.04 N, low tension—0.06 N, medium tension—0.08 N, high tension—0.1 N, and very high tension—0.24 N). Finally, the length of the muscle (in millimeters) was measured at different levels of passive tension (0.04–0.24 N). These passive tension values were chosen based on our preliminary studies. The optimal tension that generates the highest muscle force was determined by our preliminary work and confirmed in the experimental group presented in this paper. The muscle force at a given muscle length was measured in response to electrical stimulation with a train of pulses of 0.1 mA current, at a frequency of 200 pulses per second.
The SM muscle was stretched with the medium tension of 0.08 N at which the highest muscle force was consistently produced. Then, the muscle force was measured as a function of stimulation current. The intensity of stimulation current was increased starting from 0.01 mA through the level where the muscle force reached a plateau (about 0.1 mA) and continued at the supramaximal level until 0.5 mA.
To analyze the other parameters responsible for maximum force generation, a force-frequency curve was built. Two trains of stimuli (200 msec duration each, with a rest period of 20 seconds between contractions) with incrementally increasing frequencies were delivered. The stimulation frequency was increased gradually from 5 Hz (when only one pulse during the 200 ms stimulation period was given and could be used to evaluate twitch muscle force), through frequencies for which the muscle force reached a plateau (about 100 Hz) and continued to increase until 500 Hz.
Following the completion of isometric force testing, the rat was euthanized with an overdose of anesthetic. The entire SM muscle was removed and weighed (in grams).
The experiment was controlled by an Acquisition System built from a multifunction I/O National Instruments Acquisition Board (NI USB 6251, 16 bit 1.25 Ms/s, National Instruments, Austin, TX) connected to a DELL laptop with a custom written program using labVIEW 8.2 software (National Instruments, see Figure
Experimental variables included three independent variables (initial passive tension before stimulation, current and frequency of stimulation) and one dependent variable: muscle force generated during stimulation. Minimal stimulation current (when the stimulation train was set at 200 Hz), minimal stimulation frequency (when stimulation pulses were set at 0.1 mA), and optimal length of the muscle (when stimulation parameters were set at 0.1 mA and 200 Hz), which were able to produce maximal tetanic muscle contraction, were described by the means and standard deviations. The
SM muscle force was defined as the difference between maximal muscle contraction observed during electrical stimulation (with 200 ms train of pulses) and the initial tension of the muscle just before stimulation. Our goal was to establish optimal muscle length and characteristics of muscle force generated by electrical stimulation, which could be used in our further studies as a reference (muscle force generated by the muscle with an intact nerve) to evaluate the level of muscle force recovery after reinnervation. Therefore, we evaluated how muscle force depends on initial passive tension and how it changes with intensity and frequency of nerve stimulation.
Muscle force is a function of muscle length produced by an initial stretch of the muscle before electrical stimulation. The muscle was stretched with the following tensions before stimulation: very loose (0.04 N), loose (0.06 N), moderate (0.08 N), tense (0.1 N), and very tense (0.24 N). We used 0.1 mA pulses at 200 Hz. The averaged data from the whole group of rats illustrating the decrease of muscle force at different passive tensions is shown in Figure
Muscle force as a function of passive tension before stimulation. This force-tension curve was normalized by maximal force to illustrate the rate of decline of force at different passive tensions (set up just before electrical stimulation)
The muscle force was found to be the highest (mean = 0.94 N), when the muscle was initially stretched at a moderate tension (set at 0.08 N). Decreasing initial tension to “loose” (set at 0.06 N) decreased muscle force by 12% (0.82 N—statistically significant decrease
To examine the relationship between muscle force and stimulation current, we varied the current from 0 to 0.5 mA at 200 Hz trains of pulses when the muscle was stretched with moderate tension of 0.08 N, which produced optimal muscle length. Figure
Force measurements from a representative rat, showing the stimulation intensity-force relationship. (a and b) show the time course of muscle force produced by electrical stimulation of the SM nerve at eight different intensities. Note that stimulation at 0.1 mA resulted in maximal muscle contraction. (c) illustrates the outcome from these 8 measurements—the relationship between muscle force and stimulation intensity.
Muscle force as a function of stimulation current. This group average shows the density of force produced by the SM muscle and normalized by its cross-section area at different stimulation currents. Vertical bars represent the standard error of the mean. The passive tension was at a moderate level (0.08 N). The nerve was stimulated at 200 Hz.
We also analyzed how muscle force changes with regard to the frequency of stimulation pulses (from 0 to 500 Hz). We used a 200 ms train of 0.1 mA pulses. The muscle was stretched with a moderate tension (0.08 N). Figure
Illustration of muscle force measurement as a function of stimulation frequency in a representative rat. Individual muscle contractions to stimulation pulses at different frequencies are shown in red. Single stimulation pulses are indicated by green vertical lines. The muscle responded with single twitches until 25 Hz. At 50 Hz the muscle contractions were fused. With an increasing frequency of stimulation, the muscle responded with increased force, which reached a plateau at about 150 Hz.
Muscle force as a function of stimulation frequency. The group data shows the relationship between muscle force density (normalized to cross-section area) and stimulation frequency. Vertical bars represent standard error of the mean. The passive tension was at a moderate level (0.08 N). The nerve stimulation current was 0.1 mA.
A representative example in an individual rat to show the relation between muscle force and stimulation frequency at two levels of stimulation current. The passive tension was at a moderate level (0.08 N). The nerve stimulation current was 0.1 mA (continuous line) or 0.075 mA (dashed line). Muscle force is higher for a bigger current but otherwise both curves share similar characteristics with a slight decline of force for the highest frequency of stimulation.
Immediately after the experimental session, the length of the SM muscle was measured at different stretching forces, and then the muscle was removed and weighed. The average length was 25.7 mm (range 24–27 mm) at very loose tension (0.04 N), 26.8 mm at loose tension (0.06 N), 27.7 mm at moderate tension (0.08 N), 28.6 mm at tense (0.1 N), and 31.4 mm at very tense (0.24 N). The average muscle weight was 0.50 g (range 0.47–0.53 g).
This study investigated the muscle force features of the SM muscle in a rat model. We determined the correlations of muscle force (active muscle contraction to electrical stimulation) with passive tension (passive force changing muscle length) and two parameters of electrical stimulation, intensity and frequency. There are several key findings of this study. First of all, moderate muscle length/tension (28 mm/0.08 N) produced maximal muscle force (0.94 N). Second, 0.03 mA was the threshold current for initiating noticeable muscle contraction. Third, 0.2 mA was the stimulation current which produced the maximal force in the SM. Finally, the stimulation frequency that produced maximal muscle force was about 150 Hz. Taken together, in the normal rat, maximal force in the SM can be produced with moderate passive tension, 0.2 mA current, and 150 Hz frequency. These findings are important not only for better understanding the contractile properties of the rat SM muscle, but also for providing normative values which would be useful for reliably evaluating the extent of functional recovery induced by muscle reinnervation.
Muscle length is an important variable affecting active muscle force generated in response to electrical stimulation. However, establishing the optimal length of the muscle, which could produce maximal muscle force, requires lengthy investigation at many different lengths each time a new muscle is studied.
The present study shows the highest SM muscle force when the muscle is stretched with a tension of 0.08 N before stimulation (8.5% of maximal isometric tetanic force). Data from this study showed the typical “U shape” relationship between length and force in the SM muscle. Our results are consistent with the general characteristics obtained in muscle force measurements where other muscles were studied in the rat and other species [
Previous studies showed a very wide range of optimal passive tensions in different muscles and species which allow muscles to be stretched to optimal length and contract with maximal force. Celichowski et al. [
Large muscle stretching during measurement could have a detrimental impact on subsequent measurements due to stretch-induced damage. Davis et al. [
Normalized force by cross-section area in our rat SM muscle was 54 mN/mm2 (940 mN/17.5 mm2), which is lower than that obtained from the rabbit sternocleidomastoid muscle as reported by Falkenberg et al. [
Our results showed that SM muscle force in the rat grows with stimulation current until about 0.1-0.2 mA when it reaches a plateau. The threshold of stimulation current, which can generate muscle contraction with noticeable force, is about 0.03 mA. Muscle force characteristics were repeatable across different animals and therefore can be used as a normal control reference in reinnervation studies.
In many previous muscle force studies, due to the simplicity of the stimulator’s circuitry and the necessity for safety during nerve stimulation (limited maximal amplitude of stimulation), the nerves were stimulated with rectangular pulses with regulated voltage. For example, Yoshimura et al. [
Stimulation with regulated current provides more reproducible results than stimulation with regulated voltage. Regardless of electrode impedance, a reproducible electric field can be created within stimulated tissue [
On the basis of the discussed results, we selected the optimal circumstances (initial passive tension and electrical stimulation parameters) for the rat SM muscle to contract with maximal force. This maximal muscle force will be used as a target level in our further study of muscle force recovery in a denervated SM muscle where different reinnervation techniques will be compared.
Our experiments demonstrated that SM muscle force grows with the frequency of pulses until about 100–200 Hz when it reaches a plateau. A further increase of stimulation frequency produces a slight decrease of muscle force. The muscle force fusion frequency is higher than 50 Hz. The relatively high frequency of tetanic fusion might result from muscle fiber type composition. The SM muscle is a fast muscle with over 80% of type II fibers (in rats Luff [
The results of Roszek et al. point out that the commonly used procedure of establishing optimal muscle length only once at the beginning of the study with a particular frequency of stimulation might not be appropriate when the muscle force is later analyzed at different frequencies. Repetitive testing to reveal the force-length relationship at all studied frequencies seems to be more appropriate. However, the analysis of Roszek et al. also showed that the optimal muscle length changes significantly with the frequency of stimulation only when the stimulation frequency is very low—below 40 Hz. There was no significant difference in optimal muscle length between stimulation at 50 Hz and 100 Hz. Stimulation frequencies below 40 Hz were not the focus of the present study, because one of our main goals was to establish optimal stimulation parameters which would generate a frequency fused contraction of maximal force in the SM muscle. Our investigations showed that muscle contraction to a train of stimulation pulses starts to be fused at about 50 Hz (see Figure
Brooks et al. [
Falkenberg et al. [
To produce tetanic contraction, we used a 200 ms train of biphasic pulses of 0.2 ms width, typically at 200 Hz. Different frequencies of stimulation were used only when the influence of frequency of stimulation on muscle force was directly studied. This 200 ms time ensured that muscle force reached a plateau (see Figures
Shin et al. [
Integrated EMG activity is sometimes used as a nondirect method of muscle force evaluation during muscle contraction. However, gross movement of the EMG electrode caused by SM muscle contractions during obstructive breathing (as well as during electrical stimulation) might be the source of unpredictable electrical artifacts causing a lack of good correlation between integrated EMG and muscle contractions [
Optimization of the variables affecting the isometric tetanic force of the rat SM muscle resulted in a choice of the following stimulation parameters to produce maximal tetanic force: 0.08 N (moderate) passive tension before stimulation, 0.2 mA stimulation current (with biphasic 0.2 ms width pulse), and 150 Hz stimulation frequency.
This research was supported by NIH Grant 5R01DC008599 from the National Institute on Deafness and Other Communication Disorders (to Liancai Mu).
The authors are grateful to Drs. Hungxi Su and Xiaolin Zhang for their excellent technical assistance during the surgical operations and data collection.