Chronic assessment of diaphragm muscle EMG activity across motor behaviors

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.

Figure 1

Representative electromyographic (EMG) recordings and root-mean-squared (RMS) amplitude from the diaphragm muscle in an adult rat. Recordings were obtained using chronically implanted electrodes during eupnea in the same animal at day 0 (D0), D14 and D42 (A). Notice that diaphragm EMG activity occurs in bursts, reflecting rhythmic inspiration in this anesthetized animal. Minimal variability in diaphragm RMS EMG activity was observed within each recording session. Diaphragm EMG activity was also measured across the following ventilatory and non-ventilatory motor behaviors: eupnea, hypoxia-hypercapnia (10% O₂- 5% CO₂), airway occlusion and sneezing induced by intranasal capsaicin administration. Representative recordings obtained in a single animal at D42 are shown (B). Diaphragm EMG activity and RMS EMG amplitude increases significantly from ventilatory behaviors to nonventilatory behaviors, consistent with previous results (Mantilla et al., 2010).

Figure 2

Amplitude of diaphragm RMS EMG activity across different ventilatory and nonventilatory behaviors, normalized to eupnea at D0 (E0-normalization). Diaphragm EMG RMS amplitude measured at 3 different time points: D0, D14, and D42 and across 5 different motor behaviors at each time-point are shown (eupnea: ◆ hypoxia-hypercapnia: ■; deep breath: Δ; airway occlusion: ▲; sneezing: □). No significant changes in peak RMS EMG amplitude were observed over 6 weeks during eupnea, hypoxia-hypercapnia or deep breaths. During occlusion and sneezing, there was a significant effect of time: E0-normalized EMG amplitude at D14 was greater than at D0. Equivalent results are obtained when normalizing to sneezing at D0 (data not shown). All data are mean ± SE (n=8 animals; 10 electrode pairs).

Figure 3

Amplitude of diaphragm RMS EMG activity was normalized within the same time-point to that during sneezing (Snz-normaliztion; A), deep breaths (DB-normalization; B) and sustained airway occlusion (Occ-normalization; C). Notice stable measurements across behaviors (eupnea: ◆ hypoxia-hypercapnia: ■; deep breath: Δ; airway occlusion: ▲; sneezing: □). There was no significant effect of time on Snz-, DB- or Occ-normalized RMS EMG amplitude over the 6-week experimental study and no interaction between time vs. behavior. However, the CV for each behavior was reduced in DB- and Occ-normalized compared to Snz-normalized data (see text for details). All data are mean ± SE (n=8 animals; 16 electrode pairs).

Figure 4

Representative plethysmographic traces measured at D0 and D42 in an anesthetized rat during eupnea. Traces show box flow measurements (arbitrary units, a.u.) and calculated tidal volumes. Measurements were performed immediately before EMG recordings (see text for details). Notice increase in tidal volume at the end of the 6-week experimental period, which was proportional to changes in body weight.