Non-invasive assessment of muscle injury in healthy and dystrophic animals with electrical impedance myography

Abstract

Introduction

Dystrophic muscle is particularly susceptible to eccentric contraction-induced injury. We tested the hypothesis that electrical impedance myography (EIM) can detect injury induced by maximal-force lengthening contractions.

Methods

We induced injury in the quadriceps of wild type (WT) and dystrophic (mdx) mice with eccentric contractions using an established model.

Results

mdx quadriceps had significantly larger losses in peak twitch and tetany compared to losses in WT quadriceps. Injured muscle showed a significant increase in EIM characteristic frequency in both WT (177 ± 7.7 %) and mdx (167 ± 7.8 %) quadriceps. EIM also revealed decreased extracellular resistance for both WT and mdx quadriceps following injury.

Discussion

Our results show overall agreement between muscle function and EIM measurements of injured muscle, indicating that EIM is a viable tool to assess injury in dystrophic muscle.

Introduction

Duchenne muscular dystrophy (DMD) is an X-linked myopathy characterized clinically by severe, progressive, and irreversible muscle wasting, and loss of muscular function.^1–3 This disease is due to the lack of dystrophin, a large membrane-associated protein expressed in striated muscle and localized to the inner surface of the sarcolemma.^{4, 5} Dystrophin is part of the dystrophin-associated glycoprotein complex, which connects the internal cytoskeleton of the muscle fiber to the extracellular matrix. The mdx mouse, the most common animal model for DMD, also lacks dystrophin and shares similar muscle pathology to that found in DMD patients.

A number of potential medical treatments have been the focus of recent clinical trials in DMD (clinicaltrials.gov NCT02255552, NCT02257489), but only a small handful of studies have examined the potential benefit of exercise in boys with this disorder.^6–11 Unfortunately, these exercise studies are limited by inconsistency in study parameters, such as intensity, frequency, duration, and mode of exercise, which makes comparisons of outcomes difficult.^12–14 Further complicating matters, there are no easily applied biomarkers or outcome markers in this population to assess the effects of therapy outside of the 6-minute walk test.¹⁵ Thus, a convenient and easily applied tool that is sensitive to disease status, the effects of therapy, and superimposed injury would be very useful when developing and studying the impact of exercise programs in DMD boys.

Muscle electrical impedance, also known as electrical impedance myography (EIM), has been introduced as a novel neuromuscular assessment method.¹⁶ Unlike conventional electrophysiological measures of muscle and nerve,¹⁷ EIM does not assess the active electrical properties of tissues; instead, it evaluates the underlying structural and passive electrical properties of muscle.^18–21 EIM works on the premise that these properties are altered in disease,¹⁶ and earlier work has demonstrated differences in the electrical material properties of healthy muscle compared to diseased muscles. To date, EIM has proven a valuable tool for detecting a variety of neuromuscular diseases both in humans^22–29 and in animal models.^30–33 EIM has also been shown to be sensitive to the effects of therapy in spinal muscular atrophy and recovery from disuse.³⁴ Whether contraction-induced injury confers additional alterations on electrical impedance characteristics of muscle is not known. If EIM can detect such superimposed injury in DMD muscle, it would suggest that impedance-based methods could serve as convenient, easily-applied biomarkers to assess both potential improvement and injury during exercise therapies.

In this study, we tested the hypothesis that EIM can detect the interstitial edema and inflammatory response to skeletal muscle injury induced by maximal-force lengthening contractions and subsequent recovery in wild type (WT) and dystrophic (mdx) mice.

Materials and Methods

Animals

All protocols were approved by the University of Maryland Institutional Animal Care and Use Committee and complied with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. We measured muscle function in 10 wild type (WT, male, 2 month old, body weight 27.4 ± 2.7 g, C57BL/10ScSnJ, Jackson Laboratory, Bar Harbor, ME) and in 10 mdx mice (male, 2 month old, body weight 27.8 ± 2.1 g, C57BL/10ScSn-DMD^mdx/J, Jackson Laboratory, Bar Harbor, ME). Experimentation was performed with the animal anesthetized under 2% inhaled isoflurane anesthesia, which has no known effect on muscle electrical current or contractile activity³⁵ or the propagation of an externally applied electrical current. Body and muscle temperature were maintained by a heating lamp. The opposite quadriceps served as a control.

In vivo injury protocol

Quadriceps in vivo injury induced by maximal lengthening contractions was performed as described using an established model.^{36, 37} With the animal placed in a supine position, the thigh and pelvis were stabilized, and the ankle was secured onto a lever arm (Fig. 1A). The axis of the knee was aligned with the axis of the stepper motor (model T8904, NMB Technologies, Chatsworth, CA), and a torque sensor was (QWFK-8M, Sensotec, Columbus, OH) used to measure torque in Newton-millimeters (Nmm). The femoral nerve was stimulated via subcutaneous needle electrodes (J05 Needle Electrode Needles, 36BTP, Jari Electrode Supply, Gilroy, CA). Proper electrode position was determined by a series of isometric twitches and by observing isolated knee extension in the anesthetized animal. A custom program based on commercial software (LabView version 8.5, National Instruments, Austin, TX) was used to synchronize contractile activation and the onset of forced knee flexion. Injury resulted from 15 forced lengthening contractions superimposed onto maximal quadriceps contractions through a 40° – 100° arc of motion spaced approximately 1 minute apart. This range of motion is similar to that used in human studies.³⁸ Maximal isometric torque was measured before lengthening contractions and 5 minutes after the last lengthening contraction, and were used to calculate force deficits. In vivo functional measures were assessed in the same animals over time before injury (n=10), and 0 h (WT n=10, mdx n=9), 24 h (WT n=3, mdx n=2) and 48 h (WT n=4, mdx n=4) after injury.

Figure 1.

Apparatuses used to measure contractility and electrical impedance myography (EIM). (A) To produce the injury, the femur was stabilized, and the ankle was attached to a motor-driven lever arm. The femoral nerve was stimulated supramaximally (causing knee extension, black arrow), while the lever arm forced the knee joint into flexion (curved arrow). To complete 1 repetition, the quadriceps was stimulated for 200 ms to induce a peak isometric contraction prior to lengthening by the lever arm. Reproduced with permission (Pratt et. al, J Physiol, 2013). (B) Photograph of setup for EIM measurements. Under anesthesia, the mouse was placed in a supine position, and the hindlimb was placed in a fixed position. The EIM electrode was placed onto the mid-belly of the quadriceps muscle. The cartoon inset shows the electrode array arrangement and connection to the EIM measuring device.

Measurements of impedance

We measured left and right longitudinal quadriceps impedance between 10 kHz and 1 MHz (EIM1103, Skulpt Inc., San Francisco, California, USA) using a surface electrode array described elsewhere (Fig. 1B).³⁰ Data were collected after the fur was clipped and all remaining fur was removed using a depilatory agent. The skin was cleaned with 0.9% saline solution to ensure good electrode contact. Quadriceps impedance was measured in the same animals over time before injury (WT n=10, mdx n=10) and 0 h (WT n=10, mdx n=10), 24 h (WT n=10, mdx n=10), and 48 h (WT n=6, mdx n=6) after injury.

Torque measurements

All torque values reported were normalized to the resting torque immediately prior to stimulation, i.e. peak torque attained during stimulation minus resting torque immediately prior to stimulation. A twitch was elicited by a single supramaximal pulse (200 μs duration) adjusted after several twitches, separated by 1 minute. Tetanic contractions were elicited by 1 ms duration pulses in 300 ms train duration with frequency between 85–100 Hz, which was optimized for each animal. The greatest peak torque attained in the twitch and tetanic experiment was taken as peak twitch torque and peak tetanic torque. The contraction time was calculated as the time from onset of torque to peak force. Half relaxation time was calculated as the elapsed time until the peak twitch torque decreased to its half value. Peak rates of torque contraction (+dT) and decline (−dT) were determined from the maximum and minimum values of the first derivative of the twitch torque, respectively. Times to +dT and −dT were calculated as the time from onset of contraction to the time of peak rate contraction and decline.

Impedance data analysis

Raw phase angle values were analyzed at 50 kHz for the injured muscle group. The averaged multi-frequency impedance of the quadriceps muscle was modeled using the impedance model proposed by Kenneth S Cole.³⁹ The model was adjusted using a weighted nonlinear least square algorithm, and their standard errors were calculated⁴⁰ using Matlab (Mathworks, Natick, MA). As the measurement frequency is changed, the Cole plot describes a circular arc of impedance.

The Cole model summarizes multi-frequency impedance dependence into a reduced set of parameters which have a direct interpretation in terms of the geometrical and (passive) electrical properties of skeletal muscle fibers⁴¹. The muscle fibers were assumed to be essentially a cylinder of radius a composed by the surface membrane represented as a parallel combination of the membrane resistance r_m and capacitance c_m and the bulk resistive properties of the intracellular r_i and extracellular media r_e. The apex of the impedance arc (i.e. peak reactance) corresponds to the characteristic frequency at which the current is evenly split between the intracellular and extracellular compartments. The greatest resistance value in the abscissa of the Cole plot is the resistance measured by the low frequencies and is by definition, r_e. The lower resistance value in the abscissa of the Cole plot is the resistance measured at high frequencies calculated as the parallel combination between r_e and r_i. The change of c_m can be calculated from the characteristic frequency assuming constant both a and r_i after injury. The membrane resistance is r_m ≫ c_m and therefore negligible. Unless otherwise noted, values are reported as specific.

Magnetic resonance imaging

Magnetic resonance imaging (MRI) studies were performed on a Bruker Biospec 7.0 Tesla 30-cm horizontal bore scanner using Paravision 5.1 software (Bruker Biospin MRI GmbH, Germany). A Bruker 4-element ¹H surface coil array was used as the receiver, and a Bruker 72 mm linear-volume coil was used as the transmitter. One mouse of each genotype (WT n=1, mdx n=1) was anesthetized in an animal induction chamber with a gas mixture of O₂ (1 L/min) and isoflurane (3%). The animal was then placed supine on a custom-made body holder bed, and the radio frequency coil was positioned and fixed with surgical tape in the region of interest on the animal leg. After the animal was moved into the center of the magnet, the isoflurane level was maintained at 1.0 to 1.5% for the remainder of the experiment. An MR-compatible small-animal monitoring and gating system (SA Instruments, Inc., New York, NY) was used to monitor animal respiration rate and body temperature. Mouse body temperature was maintained at 36 – 37° C using a warm water circulator. Three-slice (axial, mid-sagittal, and coronal) scout rapid acquisition with fast low angle shot MR imaging was used to localize the leg. High resolution T₂-weighted MRI images in the cross-sectional view between the hip joint and the knee were acquired using rapid acquisition with relaxation enhancement (RARE) sequence with TR/TE (repetition time/echo time) = 5000/32 ms, RARE factor = 8, field of view = 30 × 30 mm², matrix size = 250 × 250, slice thickness = 0. 5 mm without a gap, averages = 16, number of slices = 32.

Statistics

The Mann-Whitney test with Bonferroni correction was used to identify differences in twitch and tetanic torque parameters between genotypes before injury and 0 h, 24 h, and 48 h after injury. Dependent impedance variables were defined as the paired difference of injured and non-injured phenotypes over time points and were analyzed using linear mixed-effects regression models with random intercept term to account for correlations within measurements over multiple time-points. The time in the model was coded using 3 indicator variables for 0 h, 24 h, and 48 h post-injury with pre-injury as the reference. Similarly, the response trajectories of only injured phenotypes were analyzed using a linear mixed-effects model. A Bonferroni-Sidak approach was used to adjust for the entire set of multiple comparisons based on the models for the trajectories of paired difference (injured vs non-injured) and only injured phenotypes within each impedance variable. All statistical tests were 2-tailed, and the significance was set at P < 0.05. Statistical calculations were conducted using Prism (GraphPad Software, La Jolla, CA, USA) and SAS version 9.4 (Cary, NC). All data are presented as mean ± standard error of the mean (S.E.M.) unless otherwise noted.

Results

mdx quadriceps muscles are more susceptible to injury than healthy muscle

Since proximal muscles are affected earlier and to a greater extent in DMD ^{42, 43}, and a similar pattern has been documented in mdx mice,⁴⁴ we performed muscle contractility and EIM on the anterior thigh muscles. mdx mice are known to generate at least as much absolute force as WT mice (Table 1, peak tetany), but less “specific force” (amount of force per unit of muscle physiological cross-sectional area) as we and others have shown previously.^{45, 46} Muscles in mdx mice show severe susceptibility to injury,^{45, 47} as demonstrated in various protocols, including a high number of repetitions through a low strain (e.g., 150 reps through a 40° arc of motion), or fewer repetitions using a larger strain.^{48, 49} We used the latter (15 repetitions through 60° arc of motion) for the in vivo quadriceps eccentric injury protocol. As expected, mdx mice sustained a significantly greater drop in isometric strength (68.4 ± 4.5% loss in maximal tetanic torque) after injury compared to WT (22.1 ± 5.0% loss in maximal tetanic torque), P < 0.0001. We also include analysis of twitch torque (Table 1), which paralleled the findings of tetanic torque, with a significantly larger drop in mdx mice (66.6 ± 5.5%) than in WT (37.3 ± 6.5%), P < 0.01 (Fig. 2C and D). Although maximal isometric torque is the standard metric for measuring contractile strength, if changes in maximal twitch are valid, this provides another possible means to assess dystrophic muscles, without subjecting them to the large forces developed during maximal isometric testing.

Table 1.

Twitch and tetanic torque parameters from healthy WT and mdx mice

		PRE-INJURY			0 h			24 h			48 h
		MEAN	STD	n	MEAN	STD	n	MEAN	STD	n	MEAN	STD	n
Peak twitch (mN mm)	WT	2.17	0.78	10	1.36	0.73	10	2.64	0.45	3	2.40	0.43	4
	mdx	2.19	0.92	10	0.76	0.47	9	2.19	0.56	2	2.61	0.82	4
	p	0.950			0.065			0.600			0.829
Contraction time (ms)	WT	19.67	1.88	10	18.08	1.81	10	16.22	3.53	3	13.44	1.64	4
	mdx	19.60	2.11	10	18.24	3.61	9	22.92	2.00	2	23.88	2.25	4
	p	0.941			0.899			0.099			*
Half relaxation time (ms)	WT	14.31	2.43	10	12.54	2.72	10	14.28	1.31	3	11.66	1.16	4
	mdx	14.07	3.36	10	13.75	4.74	9	21.06	3.39	2	21.44	2.59	4
	p	0.858			0.500			0.045			*
Time to peak dT (ms)	WT	8.67	1.58	10	8.50	2.37	10	6.39	0.98	3	3.69	1.68	4
	mdx	8.85	1.23	10	9.41	4.58	9	4.00	1.41	2	9.71	0.82	4
	p	0.775			0.589			0.106			*
Peak +dT (mN mm/ms)	WT	0.19	0.08	10	0.13	0.07	10	0.27	0.08	3	0.27	0.07	4
	mdx	0.20	0.07	10	0.08	0.05	9	0.17	0.03	2	0.20	0.07	4
	p	0.703			0.131			0.200			0.189
Time to peak −dT (ms)	WT	30.37	3.26	10	27.43	3.59	10	29.56	6.55	3	22.75	2.47	4
	mdx	29.65	4.37	10	28.52	4.84	9	42.83	8.25	2	39.13	6.35	4
	p	0.683			0.581			0.135			0.003
Peak −dT (mN mm/ms)	WT	−0.10	0.04	10	−0.08	0.04	10	−0.14	4.53	3	−0.15	0.04	4
	mdx	−0.10	0.03	10	−0.04	0.02	9	−0.09	0.02	2	−0.09	0.02	4
	p	0.978			0.019			0.053			0.032
Peak tetany (mN mm)	WT	6.41	1.48	10	5.01	1.49	10	8.53	0.95	3	8.22	2.15	4
	mdx	7.16	1.59	10	2.17	0.83	9	4.78	1.23	2	7.71	1.39	4
	p	0.290			**			0.014			0.706
Peak twitch/tetany	WT	0.34	0.10	10	0.26	0.07	10	0.31	0.02	3	0.31	0.09	4
	mdx	0.30	0.10	10	0.32	0.14	9	0.45	0.04	2	0.35	0.12	4
	p	0.471			0.202			0.011			0.639

Data are shown as mean and standard deviation. Mann-Whitney test, 2-tailed, with Bonferroni correction for multiple comparisons. Statistical significance was set after correction at P < 0.001.

^*P < 0.001;

^**P < 0.0001.

The non-significant P-values reported are the actual P-vales before correction. Abbreviations: +dT, rate of torque development; −dT, rate of torque decay; EIM, electrical impedance myography; WT, wild type; mdx, dystrophic; MRI, magnetic resonance imaging

Figure 2.

Representative pre-injury and immediately after injury (0 h post-injury) tetanic (A) and twitch (C) torque waveforms of WT and mdx mice. Loss of peak tetanic (B) and twitch (D) torque immediately after injury were normalized in each WT and mdx animal to pre-injury (0% loss in torque). After 15 lengthening contractions, mdx mice had a larger loss in peak twitch and tetanic torque, 66.6 ± 5.5% and 68.4 ± 4.5%, respectively, compared to WT mice, 37.3 ± 6.5% and 22.1 ± 5.0%, respectively. Additional twitch parameters and time points post-injury are listed in Table 1. Data shown as mean ± S.E.M. Mann-Whitney test, 2-tailed. ** P < 0.01, **** P < 0.0001.

Single and multi-frequency muscle impedance values change after injury

Single-frequency changes at 50 kHz in phase angle normalized to pre-injury values show a decrease in WT and mdx mice (Fig. 3A and B) of 12.0 ± 6.1% and 14.4 ± 4.7% 0 h after injury (P < 0.0001); and 35.2 ± 9.3% and 46.0 ± 3.0% at 24 h after injury (P < 0.0001), respectively. Both WT and mdx mice had an increase in phase angle values after 48 h of recovery, and WT mice recovered faster to pre-injury values (18.3 ± 5.1%, P < 0.001) than mdx mice (30.0 ± 5.7%, P < 0.001). These changes are indicative of gross structural and morphological alterations occurring in muscle after similar large-strain injury protocols we have used previously.^{46, 50–53} We analyzed multiple frequencies and model the data using an equivalent circuit to disentangle the source(s) that cause such changes in phase angle, (Fig. 3C and D). During the first 24 h, WT and mdx arcs of impedance shrink and shift to lower resistance (R) and reactance (X) values. Forty-eight hours post-injury WT and mdx impedance recover and shift back toward R and X pre-injury values.

Figure 3.

Specific single-frequency muscle phase angle changes of WT (A) and mdx (D) mice measured at 50 kHz before injury, and 0 h, 24 h, and 48 h post-injury. Averaged multi-frequency impedance loci from the quadriceps muscles of WT (C) and mdx mice (D). During the first 24 hours, WT and mdx impedance loci shrink and shift to lower resistance (R) and reactance (X) values. Forty-eight hours post injury WT and mdx impedance loci recover and shift towards R and X values immediately after injury. Representative frequencies are shown only in the impedance locus pre-injury for illustration purposes. Data shown as mean ± S.E.M. One-way ANOVA test with repeated measures, 2-tailed. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Characteristic frequency increases after muscle injury

Control data from the non-injured right quadriceps muscle are shown in Figure 4, solid lines. The characteristic frequency in WT muscle at 0 h and 24 h after injury increases by 137.0 ± 8.3% and 182.2 ± 15.0%, P < 0.05 and P < 0.0001, respectively (Fig. 4B). In mdx mice, however, there is a significant (P < 0.0001) change only at 24 h, showing a 170.0 ± 9.1% increase (Fig. 4C). In WT and mdx mice, at 48 h post-injury, the increase in quadriceps characteristic frequency (137.1 ± 9.2% and 138.4 ± 12.3%) is not significantly different to the non-injured values, indicating recovery of muscle physiology to pre-injury values. The factors that determine the characteristic frequency will be discussed in detail.

Figure 4.

Electrical impedance before and after injury. Illustration shows the relationship between the alternating electrical current (sine-waves) and electrical circuit parameters of a cylinder-shaped muscle fiber (A). The circuit parameters are the intracellular resistance r_i; extracellular resistance r_e; membrane capacitance c_m; membrane resistance r_m; and myofiber radius a. Changes in quadriceps characteristic frequency (B, C) within 48 h after in vivo injury (dashed lines) indicate muscle recovery. At 48 h post-injury, the increase in characteristic frequency drops to 126.5 ± 7.0% (non-significant) and 133.8 ± 7.7% (P < 0.05) in WT and mdx mice, respectively. Specific extracellular (D, E) and intracellular (F, G) resistances and membrane capacitance (H, I) from the quadriceps muscles of WT and mdx mice were also measured. Lack of substantial post-injury changes in intracellular resistance and membrane capacitance, measured by high-frequency current (Fig. 4A, red sine-wave), suggest that there was no cell swelling or cell integrity disruption in WT or mdx after injury. Non-injured data from the right quadriceps are shown as control (solid lines). Data shown as mean ± S.E.M. Two-way ANOVA test, 2-tailed. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Decrease in extracellular resistance after injury indicates interstitial edema

Specific extracellular (Fig. 4D and E) and intracellular (Fig. 4F and G) resistances from the quadriceps muscles of WT and mdx mice were measured pre-injury and 0 h, 24 h, and 48 h post-injury. Muscle injury results in interstitial edema,^54–57 which is confirmed by the loss in extracellular resistance, the latter being measured by the low-frequency current flowing through the extracellular compartment (Fig. 4A, blue sine-wave). At 0 h, 24 h, and 48 h, the loss in extracellular resistance in WT mice is 28.0 ± 4.3% (P < 0.001), 45.7 ± 5.6% (P < 0.0001), and 27.0 ± 8.3% (P < 0.01), respectively. In mdx mice, however, the loss of extracellular resistance is only significant after 24 h (43.8 ± 3.7%, P < 0.001). Lack of substantial post-injury changes in intracellular resistance as measured by high-frequency current (Fig. 4A, red sine-wave) indicates minimal cell swelling in WT or mdx mice after injury (Fig. 4F and G).

Changes in membrane capacitance suggest sarcolemmal damage after injury

There was significant interaction between changes in injured and non-injured myofiber membrane permeability as measured by the membrane capacitance (Fig. 4H and I). Normalized to pre-injury values, the membrane capacitance at 0 h, 24 h, and 48 h in WT muscle is 75.5 ± 4.7% (P < 0.01), 59.6 ± 6.5% (P < 0.0001), and 74.5 ± 4.7% (p < 0.05), respectively. In mdx mice, however, the membrane capacitance is only significant after 24 h (61.9 ± 5.3%, P < 0.001) and 48 h (76.0 ± 8.6%, P < 0.05).

Non-invasive imaging confirms edema after injury

In vivo magnetic resonance imaging (MRI) was performed at the level of the thighs. The representative images from a WT and an mdx mouse show transverse (axial) sections of T₂-weighted MRI (Fig. 5). There is clearly increased signal intensity (edema) in the quadriceps on the injured side (red arrows) compared to the uninjured control side. This increase in edema over time [within 1 hour after injury (0 h) to 24 h] was present after injury in both WT and mdx mice, paralleling the findings of a decrease in extracellular resistance after injury.

Figure 5. Edema after injury.

Representative images show transverse sections of T₂-weighted MRI (magnetic resonance imaging) in a WT and an mdx mouse before and after contraction-induced injury to the thigh (F = Femur). The injured quadriceps muscle (red arrows) is easily distinguished from the non-injured quadriceps based on the increased T₂ signal (edema, white signal). Development of edema over time (0 h to 24 h) is present after injury in both WT and mdx mice. Note that in mdx muscle that, even with no injury, there are regions of hyperintensity (dotted circle) in various muscles, a characteristic finding in dystrophic muscle. The overall shape of the thigh is altered due to compression by the apparatus used to stabilize the legs.

Discussion

Skeletal muscle damage is often accompanied by a variety of clinical symptoms and signs, including localized pain, fatigue, loss of strength, and muscle tenderness. Similarly, alterations induced by damage can be readily identified with imaging and biopsy. The purpose of this work was to determine whether in vivo surface-based EIM measurements could also reveal changes in murine skeletal muscle after contraction-induced injury. We compared values pre- and post-injury in the quadriceps muscles from both WT and mdx mice. Our findings indicate that the expected finding of loss of torque after injury was accompanied by major alterations in EIM values after injury. Although the changes in EIM did not exactly parallel the changes in torque (see 0 h), these results show that it is possible to obtain meaningful information on the condition of muscle after in vivo injury using the easily applied surface EIM technique.

Standard functional experiments showed a loss in maximal isometric torque after injury, as expected from other studies.^{37, 45, 58, 59} Comparison of contractile twitch in WT and mdx mice has previously been studied,⁶⁰ but here we show a change in twitch after eccentric injury including multiple time points. Maximal tetanic force or torque is the most common measure to assess muscle strength. Here both WT and mdx mice experience a loss in maximal tetanic torque after injury (~22% and 68%, respectively) as well as a reduction in twitch torque (~37% and 65%, respectively). In terms of magnitude, it is unclear why there was a corresponding reduction in twitch only for mdx mice. This could reflect change in excitation-contraction coupling (EC coupling),^{61, 62} or be related to changes in muscle stiffness.⁶³ In any case, since maximal isometric testing generates large forces, such testing is imprudent in patients with muscular dystrophy. If the twitch response provides the same information as maximal isometric force with regard to muscle injury, it could offer a means to assess dystrophic muscles without subjecting them to the greater forces that occur with maximal isometric testing.

Single EIM phase frequency measured at 50 kHz can detect gross alterations in muscle; however, the changes in this single value are non-specific, as they do not reflect specific muscle characteristics per se. Modeling multi-frequency EIM data allows us to go a major step further and infer the specific muscle properties that were affected after injury.^{41, 64} For skeletal muscle tissue, the characteristic frequency can increase due to a reduction in the intracellular resistance, membrane capacitance, average myofiber radii, or cell volume concentration.⁶⁵ The lack of substantial changes in intracellular resistance rule out cellular swelling, thus indicating that the single-frequency changes in phase were the result of a combined effect between interstitial edema and disruption of cell structure integrity, as detected by a reduction in extracellular resistance and membrane capacitance values. These changes in muscle extracellular resistance due to edema are similar to those found in patients with fluid removal during peritoneal dialysis.⁶⁶ In fact, cell swelling would have led to a decrease in the characteristic frequency, with an increase in myofiber radii and intracellular resistance.⁴¹ In addition, the accumulation of small inflammatory cells due to injury could have also contributed to the decrease of overall cell volume fraction measured with EIM.

The pathogenic mechanisms of dystrophin loss can explain the lack of significant differences in mdx model parameters immediately after injury (0 h) as compared to baseline. In mdx muscle, the chronic inflammatory response and persistent repair processes lead to continuous muscle regeneration.^67–69 However, we detected changes in mdx model parameters after 24 h, once the eccentric injury-induced process dominated over the underlying response of disease. This would imply that if the tool were used to evaluate DMD patients who sustained stretch injury to muscle, they would need to be followed over a period and not simply immediately after injury.

Thus far, muscle strains are revealed best by T₂-weighted MRI images, which optimize contrast between injured muscles with edema (increased signal intensity) and normal uninjured muscles. The signal intensity in MRI increases substantially, and gradually reaches the peak after injury as early as 7 hours⁷⁰ or as late as 24 h⁷¹. In this study, we used in vivo MRI to evaluate changes in edema. The imaging signal intensity (white in image) continued to increase after injury (from 0 h to 24 h). This further increase has been noted in other studies and has been termed a “secondary injury”; it is due to the onset of inflammation.^72–74 The onset and duration of inflammation can vary from muscle to muscle, especially depending on the injury, and the exact timing of a secondary injury can be missed if imaging or histology is not performed hourly. The inflammation observed with MRI is seen in both WT and mdx quadriceps after injury and supports the decrease in extracellular resistance found with EIM at 24 h.

There is an important broad translational aspect to this murine-based work. Specifically, these data suggest that EIM technology can be utilized to evaluate noninvasively the development of injury after exercise protocols in boys with DMD as well as to monitor longer term muscle condition. This is critical, since exercise and physical activity continue to be considered potentially important interventions but with undetermined value. ^{3, 13} The ability to obtain valuable insights noninvasively into how muscle condition is impacted by these therapies may be important on both the individual and collective basis. It can help clarify the beneficial effects of exercise over the long term while also identifying potential short-term injurious effects. Importantly, these interventions may play roles in conjunction with potential therapies. For example, it may become valuable to non-invasively assess the impact of exercise interventions in boys treated with antisense oligonucleotides in order to ensure they lead to improvement in muscle condition rather than inadvertent worsening. Given the fact there is delayed EIM response after injury in dystrophic muscle, patients should return to the clinic for follow-up visits. In the future, however, it may become possible for them to perform daily measurements at home using a personal EIM device, and visits could be scheduled accordingly.

Previous work has shown that EIM can detect differences in WT versus mdx muscle. Our study shows that skeletal muscle injury results in significant alterations in modeled EIM parameters in WT and mdx mice. Despite the focus on dystrophic muscle in these experiments, the results suggest that EIM can be used as a biomarker to noninvasively detect alterations in skeletal muscle associated with injury in otherwise healthy muscle, further widening its future clinical applications.