Design and pilot testing of a 26-gauge impedance-electromyography needle in wild-type and ALS mice.

INTRODUCTION/AIMS: Needle impedance-electromyography (iEMG) is a diagnostic modality currently under development that combines intramuscular electrical impedance with concentric electromyography (EMG) in a single needle. We designed, manufactured, and tested a prototype iEMG needle in a cohort of wild-type (WT) and SOD1G93A amyotrophic lateral sclerosis (ALS) mice to assess its ability to record impedance and EMG data. METHODS: A new six-electrode, 26-gauge, iEMG needle was designed, manufactured and tested. Quantitative impedance and qualitative "gestalt" EMG were performed sequentially on bilateral quadriceps of 16-wk-old SOD1G93A ALS (N = 6) and WT (N = 6) mice by connecting the needle first to an impedance analyzer (with the animal at rest) and then to a standard EMG system (with the animal fully under anesthesia to measure spontaneous activity and briefly during awakening to measure voluntary activity). The needle remained in the muscle throughout the measurement period. RESULTS: EMG data were qualitatively similar to that observed with a commercially available concentric EMG needle; fibrillation potentials were observed in 84% of the ALS mice and none of the WT mice; motor unit potentials were also readily identified. Impedance data revealed significant differences in resistance, reactance, and phase values between the two groups, with ALS animals having reduced reactance and resistance values. DISCUSSION: This work demonstrates the feasibility of a single iEMG needle conforming to standard dimensions of size and function. Further progress of iEMG technology for enhanced neuromuscular diagnosis and quantification of disease status is currently in development.

Using machine learning algorithms to enhance the diagnostic performance of electrical impedance myography.

INTRODUCTION/AIMS: We assessed the classification performance of machine learning (ML) using multifrequency electrical impedance myography (EIM) values to improve upon diagnostic outcomes as compared to those based on a single EIM value. METHODS: EIM data was obtained from unilateral excised gastrocnemius in eighty diseased mice (26 D2-mdx, Duchenne muscular dystrophy model, 39 SOD1G93A ALS model, and 15 db/db, a model of obesity-induced muscle atrophy) and 33 wild-type (WT) animals. We assessed the classification performance of a ML random forest algorithm incorporating all the data (multifrequency resistance, reactance and phase values) comparing it to the 50 kHz phase value alone. RESULTS: ML outperformed the 50 kHz analysis as based on receiver-operating characteristic curves and measurement of the area under the curve (AUC). For example, comparing all diseases together versus WT from the test set outputs, the AUC was 0.52 for 50 kHz phase, but was 0.94 for the ML model. Similarly, when comparing ALS versus WT, the AUCs were 0.79 for 50 kHz phase and 0.99 for ML. DISCUSSION: Multifrequency EIM using ML improves upon classification compared to that achieved with a single-frequency value. ML approaches should be considered in all future basic and clinical diagnostic applications of EIM.

Estimating myofiber cross-sectional area and connective tissue deposition with electrical impedance myography: A study in D2-mdx mice.

INTRODUCTION: Surface electrical impedance myography (sEIM) has the potential for providing information on muscle composition and structure noninvasively. We sought to evaluate its use to predict myofiber size and connective tissue deposition in the D2-mdx model of Duchenne muscular dystrophy (DMD). METHODS: We applied a prediction algorithm, the least absolute shrinkage and selection operator, to select specific EIM measurements obtained with surface and ex vivo EIM data from D2-mdx and wild-type (WT) mice (analyzed together or separately). We assessed myofiber cross-sectional area histologically and hydroxyproline (HP), a surrogate measure for connective tissue content, biochemically. RESULTS: Using WT and D2-mdx impedance values together in the algorithm, sEIM gave average root-mean-square errors (RMSEs) of 26.6% for CSA and 45.8% for HP, which translate into mean errors of ±363 µm2 for a mean CSA of 1365 µm2 and of ±1.44 µg HP/mg muscle for a mean HP content of 3.15 µg HP/mg muscle. Stronger predictions were obtained by analyzing sEIM data from D2-mdx animals alone (RMSEs of 15.3% for CSA and 34.1% for HP content). Predictions made using ex vivo EIM data from D2-mdx animals alone were nearly equivalent to those obtained with sEIM data (RMSE of 16.59% for CSA), and slightly more accurate for HP (RMSE of 26.7%). DISCUSSION: Surface EIM combined with a predictive algorithm can provide estimates of muscle pathology comparable to values obtained using ex vivo EIM, and can be used as a surrogate measure of disease severity and progression and response to therapy.

Predicting myofiber cross-sectional area and triglyceride content with electrical impedance myography: A study in db/db mice.

BACKGROUND: Electrical impedance myography (EIM) provides insight into muscle composition and structure. We sought to evaluate its use in a mouse obesity model characterized by myofiber atrophy. METHODS: We applied a prediction algorithm, ie, the least absolute shrinkage and selection operator (LASSO), to surface, needle array, and ex vivo EIM data from db/db and wild-type mice and assessed myofiber cross-sectional area (CSA) histologically and triglyceride (TG) content biochemically. RESULTS: EIM data from all three modalities provided acceptable predictions of myofiber CSA with average root mean square error (RMSE) of 15% in CSA (ie, ±209 µm2 for a mean CSA of 1439 µm2 ) and TG content with RMSE of 30% in TG content (ie, ±7.3 nmol TG/mg muscle for a mean TG content of 25.4 nmol TG/mg muscle). CONCLUSIONS: EIM combined with a predictive algorithm provides reasonable estimates of myofiber CSA and TG content without the need for biopsy.

Assessing the therapeutic impact of resveratrol in ALS SOD1-G93A mice with electrical impedance myography.

To aid in the identification of new treatments for amyotrophic lateral sclerosis (ALS), convenient biomarkers are needed to effectively and uniformly measure drug efficacy. To this end, we assessed the effects of the nutraceutical resveratrol (RSV) on disease onset and overall survival in SOD1-G93A (ALS) mice and compared several standard biomarkers including body mass, motor score (MS), paw grip endurance (PGE), and compound motor action potential (CMAP) amplitude, with the technique of electrical impedance myography (EIM) to follow disease progression. Eighteen ALS mice (nine females, nine males) received RSV in the chow (dose: 120 mg/kg/day) starting at 8 weeks of age; 19 ALS mice (nine females, 10 males) received normal chow; 10 wild type (WT) littermates (five females, five males) fed standard chow served as controls. Biomarker assessments were performed weekly beginning at 8 weeks. No differences in either disease onset or overall survival were found between RSV-treated and untreated ALS mice of either sex; moreover, all biomarkers failed to identify any beneficial effect of RSV when administered at this dose. Therefore, for the comparative evaluation of the ability of the various biomarkers to detect the earliest symptoms of disease, data from all animals (i.e., RSV-treated and untreated ALS mice of both sexes) were combined. Of the biomarkers tested, EIM impedance values, i.e., surface EIM longitudinal phase at 50 kHz (LP 50 kHz), and CMAP amplitude showed the earliest significant changes from baseline. LP 50 kHz values showed a rate of decline equivalent to that of CMAP amplitude and correlated with both PGE and CMAP amplitude [Spearman rho = 0.806 (p = 0.004) and 0.627 (p = 0.044), respectively]. Consistent with previous work, these findings indicate that surface EIM can serve as an effective non-invasive biomarker for preclinical drug testing in rodent models of ALS.

Altered electrical properties in skeletal muscle of mice with glycogen storage disease type II.

Electrical impedance methods, including electrical impedance myography, are increasingly being used as biomarkers of muscle health since they measure passive electrical properties of muscle that alter in disease. One disorder, Pompe Disease (Glycogen storage disease type II (GSDII)), remains relatively unstudied. This disease is marked by dramatic accumulation of intracellular myofiber glycogen. Here we assessed the electrical properties of skeletal muscle in a model of GSDII, the Pompe6neo/6neo (Pompe) mouse. Ex vivo impedance measurements of gastrocnemius (GA) were obtained using a dielectric measuring cell in 30-week-old female Pompe (N = 10) and WT (N = 10) mice. Longitudinal and transverse conductivity, σ, and the relative permittivity, εr, and Cole-Cole complex resistivity parameters at 0 Hz and infinite frequency, ρo and ρ∞, respectively, and the intracellular resistivity, ρintracellular were determined from the impedance data. Glycogen content (GC) was visualized histologically and quantified biochemically. At frequencies > 1 MHz, Pompe mice demonstrated significantly decreased longitudinal and transverse conductivity, increased Cole-Cole parameters, ρo and ρo-ρ∞, and decreased ρintracellular. Changes in longitudinal conductivity and ρintracellular correlated with increased GC in Pompe animals. Ex vivo high frequency impedance measures are sensitive to alterations in intracellular myofiber features considered characteristic of GSDII, making them potentially useful measures of disease status.

Relationships between in vivo surface and ex vivo electrical impedance myography measurements in three different neuromuscular disorder mouse models.

Electrical impedance myography (EIM) using surface techniques has shown promise as a means of diagnosing and tracking disorders affecting muscle and assessing treatment efficacy. However, the relationship between such surface-obtained impedance values and pure muscle impedance values has not been established. Here we studied three groups of diseased and wild-type (WT) animals, including a Duchenne muscular dystrophy model (the D2-mdx mouse), an amyotrophic lateral sclerosis (ALS) model (the SOD1 G93A mouse), and a model of fat-related atrophy (the db/db diabetic obese mouse), performing hind limb measurements using a standard surface array and ex vivo measurements on freshly excised gastrocnemius muscle. A total of 101 animals (23 D2-mdx, 43 ALS mice, 12 db/db mice, and corresponding 30 WT mice) were studied with EIM across a frequency range of 8 kHz to 1 MHz. For both D2-mdx and ALS models, moderate strength correlations (Spearman rho values generally ranging from 0.3-0.7, depending on the impedance parameter (i.e., resistance, reactance and phase) were obtained. In these groups of animals, there was an offset in frequency with impedance values obtained at higher surface frequencies correlating more strongly to impedance values obtained at lower ex vivo frequencies. For the db/db model, correlations were comparatively weaker and strongest at very high and very low frequencies. When combining impedance data from all three disease models together, moderate correlations persisted (with maximal Spearman rho values of 0.45). These data support that surface EIM data reflect ex vivo muscle tissue EIM values to a moderate degree across several different diseases, with the highest correlations occurring in the 10-200 kHz frequency range. Understanding these relationships will prove useful for future applications of the technique of EIM in the assessment of neuromuscular disorders.

Corrigendum: Partial Weight-Bearing in Female Rats: Proof of Concept in a Martian-Gravity Analog.

[This corrects the article DOI: 10.3389/fphys.2020.00302.].

Partial Weight-Bearing in Female Rats: Proof of Concept in a Martian-Gravity Analog.

Many studies have investigated the physiological response to microgravity in both astronauts and animals. However, while space agencies have sought to deploy more women on their missions; animal models rarely include females studies or comparisons between males and females. Therefore, we exposed adult female rats to 2 weeks of partial weight-bearing at either 100% of their normal loading (PWB100) or 40% of their normal loading (PWB40), corresponding to Martian gravity-analog, and assess muscle function, force and histomorphometry. Females exposed to PWB showed an 11.62% decline in hindlimb grip force associated with an 11.84% decrease in soleus myofiber size after 14 days of exposure, while maintaining normal blood oxygenation and stress levels. This pilot study represents the first experiment designed to understand the muscular disuse associated with a partial reduction in mechanical loading in female rats, and the first step needed to develop successful mitigating strategies. NEW AND NOTEWORTHY: This research article describes the first use of quadrupedal partial weight-bearing in female rats. This study demonstrates the feasibility of partial gravity analogs in females and allows for future investigations about the impact of sex on muscle deconditioning due to reduced mechanical loading.

Intralingual and Intrapleural AAV Gene Therapy Prolongs Survival in a SOD1 ALS Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that results in death from respiratory failure. No cure exists for this devastating disease, but therapy that directly targets the respiratory system has the potential to prolong survival and improve quality of life in some cases of ALS. The objective of this study was to enhance breathing and prolong survival by suppressing superoxide dismutase 1 (SOD1) expression in respiratory motor neurons using adeno-associated virus (AAV) expressing an artificial microRNA targeting the SOD1 gene. AAV-miRSOD1 was injected in the tongue and intrapleural space of SOD1G93A mice, and repetitive respiratory and behavioral measurements were performed until the end stage. Robust silencing of SOD1 was observed in the diaphragm and tongue as well as systemically. Silencing of SOD1 prolonged survival by approximately 50 days, and it delayed weight loss and limb weakness in treated animals compared to untreated controls. Histologically, there was preservation of the neuromuscular junctions in the diaphragm as well as the number of axons in the phrenic and hypoglossal nerves. Although SOD1 suppression improved breathing and prolonged survival, it did not ameliorate the restrictive lung phenotype. Suppression of SOD1 expression in motor neurons that underlie respiratory function prolongs survival and enhances breathing until the end stage in SOD1G93A ALS mice.

Using Electrical Impedance Myography as a Biomarker of Muscle Deconditioning in Rats Exposed to Micro- and Partial-Gravity Analogs.

As astronauts prepare to undertake new extra-terrestrial missions, innovative diagnostic tools are needed to better assess muscle deconditioning during periods of weightlessness and partial gravity. Electrical impedance myography (EIM) has been used to detect muscle deconditioning in rodents exposed to microgravity during spaceflight or using the standard ground-based model of hindlimb unloading via tail suspension (HU). Here, we used EIM to assess muscle changes in animals exposed to two new models: hindlimb suspension using a pelvic harness (HLS) and a partial weight-bearing (PWB) model that mimics partial gravity (including Lunar and Martian gravities). We also used a simple needle array electrode in lieu of surface or ex vivo EIM approaches previously employed. Our HLS results confirmed earlier findings obtained after spaceflight and tail suspension. Indeed, one EIM measure (i.e., phase-slope) that was previously reported as highly sensitive, was significantly decreased after HLS (day 0: 14.60 ± 0.97, day 7: 11.03 ± 0.81, and day 14: 10.13 ± 0.55 | Deg/MHz|, p < 0.0001), and was associated with a significant decrease in muscle grip force. Although EIM parameters such as 50 kHz phase, reactance, and resistance remained variable over 14 days in PWB animals, we identified major PWB-dependent effects at 7 days. Moreover, the data at both 7 and 14 days correlated to previously observed changes in rear paw grip force using the same PWB model. In conclusion, our data suggest that EIM has the potential to serve as biomarker of muscle deconditioning during exposure to both micro- and partial- gravity during future human space exploration.

Electrical impedance myography for the detection of muscle inflammation induced by λ-carrageenan.

Electrical impedance myography (EIM) is a technique for the assessment of muscle health and composition and has been shown to be sensitive to a variety of muscle pathologies including neurogenic atrophy and connective tissue deposition. However, it has been minimally studied in pure inflammation. In this study, we sought to assess EIM sensitivity to experimental inflammation induced by the localized intramuscular injection of λ-carrageenan. A total of 91 mice underwent 1-1000 kHz EIM measurements of gastrocnemius using a needle array, followed by injection of either 0.3% λ-carrageenan in phosphate-buffered saline (PBS) or PBS alone. Animals were then remeasured with EIM at 4, 24, 48, or 72 hours and euthanized and quantitative assessment of muscle histology was performed. Parallel alterations in both 5 and 50 kHz EIM values were identified at 4 and 24 hours, including reductions in phase, reactance, and resistance. In PBS-treated animals these values normalized by 48 hours, whereas substantial reductions in phase and reactance in 5 kHz EIM values persisted at 48 and 72 hours (i.e., values of phase 72 hours post-injection were 6.51 ± 0.40 degrees for λ-carrageenan versus 8.44 ± 0.35 degrees for PBS p<0.001, n = 11 per group). The degree of basophilic area observed in muscle sections by histology correlated to the degree of phase change at these two time points (Rspearman = -0.51, p = 0.0029). Changes in low frequency EIM parameters are sensitive to the presence of inflammatory infiltrates, and have the potential of serving as a simple means of quantifying the presence and extent of muscle inflammation without the need for biopsy.