Chronic assessment of diaphragm muscle EMG activity across motor behaviors

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Abstract

The diaphragm muscle is the main inspiratory muscle in mammals. Quantitative analyses documenting the reliability of chronic diaphragm EMG recordings are lacking. Assessment of ventilatory and non-ventilatory motor behaviors may facilitate evaluating diaphragm EMG activity over time. We hypothesized that normalization of diaphragm EMG amplitude across behaviors provides stable and reliable parameters for longitudinal assessments of diaphragm activity. We found that diaphragm EMG activity shows substantial intra-animal variability over 6 weeks, with coefficient of variation (CV) for different behaviors ∼29–42%. Normalization of diaphragm EMG activity to near maximal behaviors (e.g., deep breathing) reduced intra-animal variability over time (CV  22–29%). Plethysmographic measurements of eupneic ventilation were also stable over 6 weeks (CV  13% for minute ventilation). Thus, stable and reliable measurements of diaphragm EMG activity can be obtained longitudinally using chronically implanted electrodes by examining multiple motor behaviors. By quantitatively determining the reliability of longitudinal diaphragm EMG analyses, we provide an important tool for evaluating the progression of diseases or injuries that impair ventilation.

Highlights

► Diaphragm EMG activity shows substantial variability within animals over time. ► Chronically implanted electrodes allow longitudinal assessment of diaphragm activity. ► Stable and reliable measurements can be obtained by examining multiple motor behaviors.

Introduction

As the major inspiratory pump muscle in mammals, the diaphragm muscle is of particular importance in the neuromotor control of respiration (Sieck, 1991). Many studies have examined diaphragm activity in anesthetized and freely moving animals usually by recording electromyographic (EMG) activity (Mantilla et al., 2010, Trelease et al., 1982). Assessments of ventilatory muscle activity are clearly important in evaluating the progression of and recovery from disease or injury (Mantilla and Sieck, 2003, Mantilla and Sieck, 2008, Mantilla and Sieck, 2009, Sieck and Mantilla, 2009). However, simple measures of diaphragm EMG activity (e.g., average rectified integrated (Dow et al., 2006, Dow et al., 2009) or root-mean-squared, RMS amplitude (Mantilla et al., 2010, Sieck and Fournier, 1990)) show substantial variability across animals complicating quantitative, longitudinal assessments of ventilatory activity. Furthermore, although previous techniques were reported for chronic recordings of diaphragm EMG activity in cats (Trelease et al., 1982), rabbits (Shafford et al., 2006) and guinea pigs (Chang and Harper, 1989), longitudinal, quantitative analyses documenting the reliability of such measurements are lacking.

In mammals, the diaphragm muscle is activated during ventilatory behaviors (e.g., normal resting breathing – eupnea – and exercise-induced hyperventilation) as well as during non-ventilatory behaviors associated with airway clearance (e.g., airway occlusion and sneezing) (Mantilla et al., 2010, Sieck and Fournier, 1989). Importantly, diaphragm EMG measurements reflect differences in force (Eldridge, 1975, Mantilla et al., 2010, Sieck and Fournier, 1989). Over time, a number of factors may cause variability in EMG amplitude including electrode movement, dislodgment or failure, peri-electrode tissue scarring and fibrosis as well as muscle fiber growth. Such factors may thus further complicate longitudinal assessments of ventilatory activity using EMG recordings. However, assessment of diaphragm EMG activity across ventilatory and non-ventilatory behaviors may allow comparisons across recording sessions. In particular, we recently reported that spontaneous deep breaths (“sighs”) consistently produce near maximal diaphragm activation (Mantilla et al., 2010). Thus, we hypothesized that normalization of diaphragm RMS EMG amplitude with respect to deep breaths provides a stable and reliable parameter for the longitudinal assessments of diaphragm muscle activity.

Section snippets

Animals

Eleven Sprague-Dawley adult male rats (3 months of age) with an initial body weight of ∼300 g were used in the present study. All procedures were in accordance with the American Physiological Society Animal Care Guidelines and were approved by the Institutional Animal Care and Use Committee. Animals were anesthetized with a mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg) via intramuscular injection for all surgical procedures and experimental measurements.

Intramuscular diaphragm EMG

Procedures for electrode placement

Results

EMG electrodes were successfully implanted in all 11 animals. In most animals, electrodes in either the right or left side required re-externalization due to animal biting/pulling. These electrode pairs could no longer be accessed externally, but all of them were available for diaphragm EMG recordings following surgical re-exposure. However, these recordings were censored from subsequent longitudinal analyses in order to eliminate any effect of a possible change in electrode configuration

Discussion

In this 6-week study, we found that diaphragm EMG amplitude shows substantial intra-animal variability across time (CV: 29–42% for the different ventilatory and non-ventilatory behaviors). Plethysmographic measurements of ventilatory parameters were stable during eupnea (CV: 13% for minute ventilation). Consistent with our hypothesis, normalization of diaphragm EMG activity to near maximal behaviors such as deep breathing results in much reduced intra-animal variability over time (22–29%).

Conclusions

Electromyography is a common tool used to assess muscle activation in studies examining neuromotor control of breathing. Chronic recordings of diaphragm EMG activity are potentially useful in evaluating the progression of or recovery from pathological conditions or injury resulting in impaired ventilation. However, substantial variability across animals permits only qualitative analyses over time when using raw measures of EMG amplitude (e.g., RMS). By examining diaphragm EMG activity across

Acknowledgements

This study was supported by funding from the National Institutes of Health (grants AR051173 and HL096750), the Paralyzed Veterans of America Research Foundation and Mayo Clinic.

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