Chronic assessment of diaphragm muscle EMG activity across motor behaviors
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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2018, Experimental NeurologyCitation Excerpt :Sighs were often followed by a post-sigh apnea; (4) Tracheal occlusion (TOCC): measured during the last 5 s of ~ 40s sustained TOCC. Spontaneous sighs and the response to TOCC were monitored as an indication of near-maximal respiratory muscle activation (Mantilla et al., 2011; Seven et al., 2014). After experiments, 6 rats per group were perfused and tissues fixed with 4% paraformaldehyde in 0.1 M phosphate buffered saline and harvested (Lovett-Barr et al., 2012).
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2017, Respiratory Physiology and NeurobiologyCitation Excerpt :All RMS EMG measurements were normalized to the pre-injury sigh RMSpeak for each animal. We have previously demonstrated that normalizing RMS values to near maximal behaviors such as sigh and sneeze reduces inter-animal variability over time (Mantilla et al., 2011). In addition, the tension-time index of the diaphragm was used as estimate of the efficiency of diaphragm activation before, immediately after, and 14 days after DNV was derived from the Pdi and EMG (Bellemare and Grassino, 1982).