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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix

Non-technical summary  Normal breathing is controlled by specialized neurons in the central nervous system including the brainstem and spinal cord. Signals generated in the brainstem and transmitted to the major inspiratory muscles, including the diaphragm and intercostal muscles, are necessary to sustain life. However, we show that the specific pattern of intercostal muscle activation during breathing does not require input from the brainstem. In other words, the neural circuitry controlling this pattern of activation exists within the spinal cord. This knowledge furthers our understanding of the mechanisms that control breathing and has implications for patients with certain disease states such as cervical spinal cord injury.

Abstract  In contrast to previous methods of electrical stimulation of the inspiratory muscles, high frequency spinal cord stimulation (HF-SCS) results in more physiological activation of these muscles. The spatial distribution of activation to the external intercostal muscles by this method is unknown. In anaesthetized dogs, multiunit and single motor unit (SMU) EMG activity was monitored in the dorsal portion of the 3rd, 5th and 7th interspaces and ventral portion of the 3rd interspace during spontaneous breathing and HF-SCS following C2 spinal section. Stimulus amplitude during HF-SCS was adjusted such that inspired volumes matched spontaneous breathing (Protocol 1). During HF-SCS, mean peak SMU firing frequency was highest in the 3rd interspace (dorsal) (18.8 ± 0.3 Hz) and significantly lower in the 3rd interspace (ventral) (12.2 ± 0.2 Hz) and 5th interspace (dorsal) (15.3 ± 0.3 Hz) (P < 0.05 for each comparison). Similar rostrocaudal and dorsoventral gradients of activity were observed during spontaneous breathing prior to C2 section. No significant activity was observed in the 7th interspace during either spontaneous breathing or HF-SCS. Since peak discharge frequencies of the SMUs were higher and rib cage movement greater during HF-SCS compared to spontaneous breathing, stimulus amplitude during HF-SCS was adjusted such that rib cage movement matched (Protocol 2). Under these conditions, mean peak SMU frequencies and rostrocaudal and dorsoventral gradients of activity during HF-SCS were not significantly different compared to spontaneous breathing. These results indicate that (a) the topographic pattern of electrical activation of the external intercostal muscles during HF-SCS is similar to that occurring during spontaneous breathing and (b) differential descending synaptic input from supraspinal centres is not a required component of the differential spatial distribution of external intercostal muscle activation. HF-SCS may provide a more physiological method of inspiratory muscle pacing.


Abbreviations 
HF-SCS

high frequency spinal cord stimulation

SMU

single motor unit

To

time of onset

Ti

inspiratory time

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix

Compared to previous methods of electrical stimulation of the inspiratory muscles (Glenn, 1980; Glenn et al. 1980, 1984; DiMarco et al. 1987, 1994; DiMarco, 2005), recent studies indicate that these muscles can be electrically activated in a more physiological fashion by the application of high frequency spinal cord stimulation (HF-SCS) (DiMarco & Kowalski, 2009, 2010). This technique involves stimulation of spinal tracts located on the ventral surface of the spinal cord which synapse with the inspiratory motoneuron pools. This method allows for processing of the stimulus within the motoneuron pools and activation of both the diaphragm and inspiratory intercostal muscles at physiological firing frequencies (DiMarco & Kowalski, 2009, 2010). Compared to currently available methods (DiMarco, 2005, 2006), therefore, HF-SCS holds promise as a new, and possibly more effective, method of inspiratory muscle pacing in patients with ventilator dependent tetraplegia.

While activating the external intercostal muscles at physiological firing frequencies, the specific pattern of activation of the various external intercostal muscles during HF-SCS is unknown. During spontaneous breathing, the distribution of neural drive to the external intercostal muscles in both humans (De Troyer et al. 2003; Saboisky et al. 2007) and animals (Kirkwood et al. 1984; Greer & Martin, 1990; Legrand & De Troyer, 1999) occurs along dorsoventral and rostrocaudal gradients. More specifically, inspiratory drive to the external intercostals is greater in the rostral compared to the caudal segments and greater in the dorsal compared to the ventral portions of the rib cage. The significance of this pattern of activation lies in the fact that the distribution of motor drive to the individual intercostal muscles closely matches their mechanical advantage (De Troyer et al. 2005).

The spatial distribution of external intercostal muscle activation may result from differences in synaptic inputs from central sources. Connections between inspiratory bulbospinal neurons and external intercostal motoneurons, however, do not differ among the upper thoracic segments (T2–T9). Moreover, the magnitude of total average depolarization generated by direct connections from inspiratory bulbospinal neurons to external intercostal muscles is weak, making this mechanism unlikely (De Troyer et al. 2005). In contrast, it appears that external intercostal motoneurons receive strong synaptic inputs from spinal interneurons (Kirkwood et al. 1988). We hypothesized, therefore, that the neural circuitry responsible for the specific pattern of external intercostal muscle activation resides at the level of the spinal cord and does not require differential supraspinal input. In the present investigation, therefore, we monitored the spatial distribution of external intercostal muscle activation during HF-SCS in a C2 spinal section preparation by measuring multiunit and single motor unit activity. We demonstrate that HF-SCS results in a pattern of inspiratory intercostal muscle activation which is similar to that occurring during spontaneous breathing.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix

Ethical approval

Studies were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University, Cleveland, OH, USA.

Studies were performed on seven dogs (mean weight 16.9 ± 0.5 kg). Animals were initially anaesthetized with pentobarbital sodium (25 mg kg−1) given intravenously. Additional doses were given, as required, based upon the status of corneal reflexes and response to noxious stimuli, both of which were suppressed. After completion of the experiments, animals were killed with pentobarbital sodium (100 mg kg−1 given i.v.).

In each animal, a large bore cuffed endotracheal tube (10 mm ID) was sutured into the trachea in the mid cervical region. A femoral vein catheter was placed to administer fluid and supplemental anaesthesia. A femoral arterial catheter was placed to monitor blood pressure and heart rate (Waveline Pro multi-function monitor, DRE Inc., Louisville KY, USA). A heating blanket (Harvard Apparatus, Holliston, MA, USA) was used to maintain body temperature at 38 ± 0.5°C. End-tidal inline image was monitored at the trachea and oxygen saturation from the earlobe (Waveline Pro multi-function monitor). Tidal volume was recorded by electrical integration of the flow signal from a pneumotachograph (Series 3700, Hans Rudolph Inc., Shawnee, KS, USA).

Following a laminectomy at the T4 level, an eight-plate lead with 4 mm contacts (Carefusion, San Diego, CA, USA) was positioned under direct vision on the ventral surface of the spinal cord and advanced to the T2 level (as previously described) (DiMarco & Kowalski, 2009, 2010). A Grass square-wave pulse stimulator (model S88, Grass Technologies, West Warwick, RI, USA) equipped with a stimulus isolation unit (SIU5, Grass Technologies) was used to provide electrical stimulation. Stimulus train duration was set at 1.3 s since this duration approximated that occurring during spontaneous breathing.

Respiratory displacements of the chest wall were monitored by inductance plethysmography (Braebon Medical Corporation, Kanata, ON, Canada). The effort belts were placed around the rib cage at the lower border of the sternum and around the abdomen at the level of the umbilicus, respectively. Gains of the two signals were adjusted during spontaneous efforts following airway occlusion.

Bipolar Teflon-coated, stainless steel fine-wire electrodes, uninsulated at their terminal ∼5 mm, were used to assess multiunit inspiratory muscle EMG recordings of the external intercostal muscles (dorsal and ventral portions of the 3rd interspace and dorsal portions of the 5th and 7th interspaces). Inspiratory muscle activation was further characterized by single motor unit (SMU) recordings. SMU recordings were made using Teflon-coated stainless steel electrodes with an uninsulated portion of ∼1 mm, to provide greater selectivity. EMG potentials were amplified (1000–10,000 times) and filtered (50 Hz to 5.0 kHz) (model BMA-830, CWE, Inc., Ardomore, PA, USA). All recordings were monitored and stored on an eight-channel data-acquisition recorder (model Dash 8X, Astro-Med, Inc., West Warwick, RI, USA) for subsequent analysis (AstroView X, Data Review Software, AstroMed).

Following measurements obtained during spontaneous breathing, the cervical spinal cord was sectioned at the C2 level in each animal using watchmaker forceps. A hook forceps was passed across the area of transection to verify complete section.

Protocol 1 (n= 7) EMG recording electrodes were positioned in each of the external intercostal muscles to assess their pattern of activation. Multiunit and SMU measurements of each muscle were initially taken during spontaneous breathing. Following C2 section, EMG measurements were also made during HF-SCS (300 Hz; 0.2 ms pulse width) at the T2 level; stimulus amplitude was adjusted to approximate the magnitude of inspired volume observed during spontaneous breathing (mean stimulus amplitude of 0.47 ± 0.05 mA). Five to ten sites were sampled from each muscle for SMU recordings. At each site, one to four SMUs were distinguished on the basis of their morphology. Several breaths were analysed at each site.

Protocol 2 (n= 4) Since preliminary trials indicated that the rib cage contribution to inspired volume was greater during HF-SCS compared to spontaneous breathing, stimulus amplitude was adjusted to approximate the magnitude of rib cage movement observed during spontaneous breathing (mean stimulus amplitude of 0.24 ± 0.04 mA). This additional manoeuvre was performed to determine the distribution of inspiratory activity to the external intercostal muscles under conditions in which activation was more comparable to spontaneous breathing. Otherwise, the protocol procedure was the same as in Protocol 1.

Data analysis During spontaneous breathing, the time of onset of multiunit EMG of the external intercostal muscles (To) was determined relative to the onset of inspiratory flow; during HF-SCS, the onset of multiunit EMG of these same muscles was determined relative to the onset of the stimulus pulse. Inspiratory time (Ti) was determined from the flow signal. As an additional index of the inspiratory timing of EMG signals, To was also expressed as a percentage of Ti. Values were averaged over five consecutive breaths.

Single motor unit analyses included determination of mean peak discharge frequencies, which were assessed over several consecutive breaths. The discharge frequency of each SMU during spontaneous breathing and HF-SCS was plotted against the number of motor units recorded.

Comparisons were made, where applicable, using one-way ANOVA and post hoc Newman–Keuls tests. A P value < 0.05 was taken as statistically significant. Data are reported as means ±s.e.m.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix

Protocol 1 (n= 7)

Mean inspired volumes during spontaneous breathing and HF-SCS, during which time EMG measurements were made, are presented in Table 1. There were no significant differences in inspired volumes measured during spontaneous breathing compared to those made during HF-SCS. While the magnitude of inspired volume is similar under both conditions, rib cage contribution to inspired volume was significantly greater during HF-SCS compared to spontaneous breathing (53.2 vs. 31.2%, P < 0.05).

Table 1.  Mean inspired volume and rib cage contribution to inspired volume during spontaneous breathing and HF-SCS
 UnitTidal volume matching (n= 7)Rib cage contribution matching (n= 4)
Spontaneous breathingHF-SCSSpontaneous breathingHF-SCS
  1. *P < 0.05 compared to spontaneous breathing.

Tidal volumeml249.6 ± 9.0251.1 ± 9.2241.8 ± 8.7115.6 ± 20.9*
Volume per unit weightml kg−114.8 ± 0.114.9 ± 0.314.8 ± 0.26.9 ± 0.9*
Rib cage contributionml74.4 ± 7.2128.8 ± 17.0*72.1 ± 8.876.3 ± 8.0
Rib cage contribution%31.2 ± 2.953.2 ± 7.5*29.8 ± 3.356.1 ± 6.1*

The pattern of multiunit EMG activity of the external intercostal muscles during spontaneous breathing (left panel) and during HF-SCS with matching of inspired volume (middle panel) is shown for one animal in Fig. 1. As shown in the lower portion of the figure, rib cage contribution to inspired volume was larger during HF-SCS. As with spontaneous breathing, HF- SCS results in an asynchronous pattern of intercostal muscle activation at each recording site. During both spontaneous breathing and HF-SCS, the intercostal muscles of the 3rd interspace (dorsal and ventral) and 5th interspace (dorsal) were electrically active. No electrical activity was detectable in the 7th interspace space (dorsal) during either spontaneous breathing or HF-SCS. During spontaneous breathing, the onset of intercostal EMG activity was delayed relative to the onset of inspiratory flow. Moreover, the onsets of intercostal activity in the ventral portion of the 3rd interspace and dorsal portion of the 5th interspace were delayed relative the onset of activity in the dorsal portion of the 3rd interspace.

image

Figure 1. Multiunit EMG recordings form the external intercostal muscles during spontaneous breathing and high frequency spinal cord stimulation (HF-SCS) in one animal From top to bottom, tracings represent multiunit EMG recordings of the external intercostal muscles from the dorsal portion of the 3rd interspace, ventral portion of the 3rd interspace, dorsal portion of the 5th interspace and dorsal portion of the 7th interspace; rib cage movement; airflow; and inspired volume. Recordings were obtained during spontaneous breathing (left panel) and HF-SCS with matching of inspired volume to spontaneous breathing (middle panel) and during HF-SCS with matching of rib cage movement to spontaneous breathing (right panel). See text for further explanation.

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Mean delays in timing during spontaneous breathing (expressed relative to the onset of inspiratory flow and percentage of inspiratory time) and HF-SCS (relative to stimulus onset) are presented in Table 2. During spontaneous breathing, the onset of external intercostal EMG in the dorsal portion of the 3rd interspace occurred with a mean delay of 307 ± 18 ms from the onset of inspiratory flow and To/Ti of 21 ± 1%. In addition, the onset of intercostal activity in the ventral portion of the 3rd interspace and dorsal portion of the 5th interspace were significantly delayed relative to the onset of intercostal activity in the dorsal portion of the 3rd interspace, (P < 0.05 for each). During HF-SCS, the onset of external intercostal EMG in the dorsal portion of the 3rd interspace occurred following a delay of 2.45 ± 0.04 ms from the onset of stimulation. In Protocol 1, the onset of activity in the ventral portion of the 3rd interspace and dorsal portion of the 5th interspace were significantly delayed relative to the onset of intercostal activity in the dorsal portion of the 3rd interspace (P < 0.05). The onset of inspiratory flow during HF-SCS expressed relative to the onset of the stimulus occurred with a mean delay of 107 ± 4 ms. In Protocol 2, the same pattern of delays was observed although their magnitude was somewhat greater (Table 2).

Table 2.  Delays in onset of external intercostal EMG activity during spontaneous breathing and HF-SCS
 Spontaneous breathing (n= 7)HF-SCS
Delay (ms)To/Ti (%)Tidal volume matching (n= 7) Delay (ms)Rib cage contribution matching (n= 4) Delay (ms)
  1. *P < 0.05 compared to 3rd interspace dorsal during spontaneous breathing and HF-SCS, respectively. †P < 0.05 compared to HF-SCS with tidal volume matching

3rd interspace dorsal307 ± 1821 ± 12.45 ± 0.043.81 ± 0.28†
3rd interspace ventral604 ± 27*42 ± 2*5.53 ± 0.55*8.25 ± 1.51*†
5th interspace dorsal489 ± 28*34 ± 2*2.88 ± 0.07*11.48 ± 0.33*†

Single motor unit activities of the external intercostal muscles, during spontaneous breathing and HF-SCS, are provided for one animal in Fig. 2. Action potentials from SMUs are superimposed to the right of each tracing for each muscle confirming their similar morphology. Instantaneous firing frequencies of the motor units identified in each muscle are provided below each tracing. With matching of inspired volumes (middle panel), the firing frequencies during HF-SCS for each of the external intercostal muscle EMG recordings are somewhat higher than those occurring during spontaneous breathing. As shown in the lower portion of the figure (left and middle panels), these firing frequencies are associated with a greater rib cage contribution to inspired volume (Table 1).

image

Figure 2. Single motor unit recordings from the external intercostal muscles during spontaneous breathing and HF-SCS in one animal From top to bottom, tracings represent single motor unit EMG recordings of the external intercostal muscles from the dorsal portion of the 3rd interspace, ventral portion of the 3rd interspace, dorsal portion of the 5th interspace; rib cage movement; and inspired volume. Recordings were obtained during spontaneous breathing (left panel) and HF-SCS with matching of inspired volume to that observed during spontaneous breathing (middle panel) and during HF-SCS with matching of rib cage movement to that observed during spontaneous breathing (right panel). All the action potentials from each motor unit are superimposed on the right of each tracing. See text for further explanation.

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Histograms of the discharge frequencies of all single motor units identified in the intercostal muscles in the 3rd (dorsal and ventral) and 5th dorsal interspaces are provided in Fig. 3. Mean peak discharge frequencies of the SMUs in the dorsal portion of the 3rd interspace were 12.7 ± 0.2 Hz during spontaneous breathing and 18.8 ± 0.3 Hz during HF-SCS (P < 0.05). Mean peak discharge frequencies of the SMUs in the ventral portion of the 3rd interspace and dorsal portion of the 5th interspace were significantly lower than that of the dorsal portion of the 3rd interspace, during both spontaneous breathing and HF-SCS (P < 0.05 for each comparison).

image

Figure 3. Histograms of the discharge frequencies of all single motor units identified in the dorsal portion of the 3rd interspace (upper panel), ventral portion of the 3rd interspace (middle panel) and dorsal portion of the 5th interspace (lower panel) during spontaneous breathing (grey bars) and during HF-SCS (black bars) Bin width, 1 Hz. Recordings were obtained under conditions in which inspired volume during HF-SCS was matched to inspired volumes recorded during spontaneous breathing by adjustment of stimulus amplitude. See text for further explanation.

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Expressed as a percentage of the mean peak firing frequencies of the SMUs in the dorsal portion of the 3rd interspace under each condition, the mean peak firing frequencies of the SMUs in the ventral portion of the 3rd and dorsal portion of the 5th interspaces during spontaneous breathing and HF-SCS were not significantly different (Table 3).

Table 3.  Mean peak firing frequencies of SMU activity of external intercostal muscles during spontaneous breathing and HF-SCS
 Tidal volume matching (n= 7)Rib cage contribution matching (n= 4)
Spontaneous breathing (Hz)HF-SCS (Hz)Spontaneous breathing (Hz)HF-SCS (Hz)
  1. Values in parentheses are percentages of mean peak SMU of the 3rd interspace dorsal. *P < 0.05 compared to spontaneous breathing. †P < 0.05 compared to 3rd interspace dorsal during spontaneous breathing and HF-SCS.

3rd interspace dorsal12.7 ± 0.218.8 ± 0.3*12.4 ± 0.212.2 ± 0.3
3rd interspace ventral9.0 ± 0.2†12.2 ± 0.2*†8.8 ± 0.3†8.9 ± 0.1†
(70.8 ± 3.2%)†(64.9 ± 1.1%)*†(69.3 ± 5.4%)†(71.1 ± 2.5%)†
5th interspace dorsal10.9 ± 0.2†15.3 ± 0.3*11.0 ± 0.3†10.2 ± 0.3†
(85.2 ± 2.9%)†(80.3 ± 1.8%)*†(85.9 ± 5.0%)†(85.2 ± 5.1%)†

Protocol 2

Mean inspired volumes during spontaneous breathing and HF-SCS during which EMG measurements were made are presented in Table 1. Under these conditions, the absolute rib cage contribution to inspired volumes during HF-SCS was well matched and not significantly different from that occurring during spontaneous breathing. Inspired volumes during spontaneous breathing, however, were significantly greater than those occurring during HF-SCS (P < 0.05).

The pattern of multi-unit EMG is shown for one animal in Fig. 1 (right panel). Data were obtained from the same animal in which inspired volumes were matched with spontaneous breathing (middle panel). With reduction in stimulus amplitude, EMG activity was reduced. The onset of inspiratory flow during HF-SCS relative to stimulus onset was 201 ± 16 ms. Single motor unit activities during spontaneous breathing and HF-SCS are shown for one animal in Fig. 2. In this example, the same motor units recorded during HF-SCS under conditions in which inspired volume was matched during spontaneous breathing (middle panel) was also monitored under conditions in which rib cage volume was matched with spontaneous breathing (right panel). Under these latter conditions, firing frequencies of motor units appeared lower for each portion of the 3rd and the 5th external intercostal muscles when compared to the middle panel, but similar to spontaneous breathing (left panel).

Histograms of the discharge frequencies of all motor units identified in the intercostal muscles in the 3rd (dorsal and ventral) and dorsal portion of the 5th interspace are provided in Fig. 4. Mean peak discharge frequencies of the SMUs in the dorsal portion of the 3rd interspace were 12.4 ± 0.2 during spontaneous breathing and 12.2 ± 0.3 Hz during HF-SCS (NS). Mean peak discharge frequencies of the SMUs in the ventral portion of the 3rd interspace and dorsal portion of the 5th interspace were significantly lower than that of the dorsal portion of the 3rd interspace, during both spontaneous breathing and HF-SCS (P < 0.05 for each comparison).

image

Figure 4. Histograms of the discharge frequencies of all single motor units identified in the dorsal portion of the 3rd interspace (upper panel), ventral portion of the 3rd interspace (middle panel) and dorsal portion of the 5th interspace (lower panel) during spontaneous breathing (grey bars) and during HF-SCS (black bars) Bin width, 1 Hz. Recordings were obtained under conditions in which rib cage movement during HF-SCS was matched to inspired volumes recorded during spontaneous breathing by adjustment of stimulus amplitude. See text for further explanation.

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Expressed as a percentage of the mean peak firing frequencies of the SMUs in the dorsal portion of the 3rd interspace under each condition, mean peak firing frequencies of the SMUs in the ventral portion of the 3rd and dorsal portion of the 5th interspaces during spontaneous breathing and HF-SCS were not significantly different (Table 3). Moreover, the percentage reductions in firing frequencies during both spontaneous breathing and HF-SCS were not significantly different compared to Protocol 1 in which inspired volumes were matched.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix

The results of this study demonstrate that the spatial distribution of inspiratory activity to the external intercostal muscles of the upper rib cage during HF-SCS is similar to that occurring during spontaneous breathing. When compared at the same inspired volumes, mean peak SMU discharge frequencies of the external intercostal muscles during HF-SCS were significantly higher than those occurring during spontaneous breathing. Despite these differences, the relative spatial distribution of inspiratory activity, i.e. electrical activation of the dorsal portion of the 3rd interspace being greater than both the ventral portion of the 3rd interspace and dorsal portion of the 5th interspace, was preserved in the same proportion during HF-SCS. Moreover, when stimulus amplitude was adjusted such that the absolute rib cage contribution to inspired volume during HF-SCS matched that occurring during spontaneous breathing, the spatial distribution of electrical activation was also maintained in the same proportion. These observations indicate that the dorsoventral and rostrocaudal gradients of external intercostal muscle activation during HF-SCS are similar to those occurring during spontaneous breathing.

Comparison to previous studies

The pattern of external intercostal muscle activation has been previously evaluated in both dog (Kirkwood et al. 1984; Greer & Martin, 1990; Legrand & De Troyer, 1999) and human studies (De Troyer et al. 2003) and is qualitatively similar to the results presented here. In animal experiments (Legrand & De Troyer, 1999), the peak heights of integrated EMG activities during spontaneous breathing were compared to the magnitude of the EMG signal during tetanic stimulation of the external intercostal nerves in their respective interspaces in an attempt to provide a quantitative assessment of the degree of muscle activation. The magnitude of inspiratory activity recorded from the dorsal portion of the external intercostal muscles decreased progressively from the 2nd to the 6th interspace. External intercostal inspiratory activity also decreased from the dorsal to the ventral portion of the 4th interspace. Moreover, these gradients were maintained during breathing stimulated with the application of mechanical loads and hyperoxic hypercapnia. Utilizing measurements of single motor unit activity, similar gradients were also demonstrated in subsequent human trials by De Troyer et al. (2003). These investigations demonstrate that the mean discharge frequency of SMUs in the dorsal portion of the 3rd interspace was significantly greater than the discharge frequency of motor units in both the ventral portion of the 3rd interspace and the dorsal portion of the 5th interspace. Activity in the 7th interspace was usually absent. The onsets of multiunit activity in the ventral portion of the 3rd interspace and dorsal portion of the 5th interspace were also delayed relative to the onset of activity in the dorsal portion of the 3rd interspace.

While the distribution of inspiratory activity to the external intercostal muscles was found to be similar in the present investigation, the degree of activation was significantly greater during HF-SCS compared to spontaneous breathing. This was not entirely surprising given the fact that, when inspired volumes during HF-SCS were matched to spontaneous breathing, rib cage contribution to inspired volume was also significantly greater. When absolute rib cage contribution to inspired volume during HF-SCS was matched to that occurring during spontaneous breathing, mean peak discharge frequencies were not significantly different when compared to those occurring during spontaneous breathing. Similar to previous studies (De Troyer et al. 2003), we also found that the onset of activation of the external intercostal muscles varies substantially with interspace during spontaneous breathing, i.e. onset of activity in the dorsal portion of the 3rd interspace had an earlier onset compared to the ventral portion of the 3rd interspace and dorsal portion of the 5th interspace. However, the onset of intercostal activity during HF-SCS in the two regions of the 3rd interspace and dorsal region of the 5th interspace occurred with very brief delays following the stimulus pulse. Assuming a single synaptic delay at the level of the motoneurons, and another at the neuromuscular junction, and delay related to the distance between the stimulating and recording electrodes, total delays of 2.15, 3.29 and 2.89 ms for the dorsal and ventral portions of the 3rd interspace and dorsal portion of the 5th interspace, respectively, can be predicted. While the actual delay of 2.45 and 2.88 ms for the dorsal portions of the 3rd and 5th interspaces (Protocol 1), approximates the predicted values, that of the ventral portion of the 3rd interspace (5.53 ms) exceeds the predicted value by more than 2 ms. Activation of the latter muscle portion, therefore, suggests additional synaptic delays, possibly involving interneurons in the spinal cord. With Protocol 2, delays were significantly greater than Protocol 1. While the mechanism of this observation is unknown, reductions in stimulus current are likely to have reduced the degree of spinal cord pathway activation and, in turn, the degree of EPSPs of motoneuron pool activation resulting in further delays in the onset of activation.

Physiological implications

The mechanism by which the external intercostal muscles may be differentially activated is unknown. Since the current studies were performed in a C2 section preparation, however, these results clearly demonstrate that differential descending synaptic input from supraspinal centres is not a required component of the observed responses. Stated differently, the neural circuitry responsible for the unique distribution of neural drive to the external intercostal muscles is present at a spinal cord level.

Potential mechanisms operative at the level of the spinal cord which could mediate differential external intercostal activation include (a) non-uniform spatial distribution of mechanoreceptors within portions of the external intercostal muscles resulting in differential activation of the α-motoneuron pools, (b) differences in the intrinsic properties of external intercostal α-motoneurons (De Troyer et al. 2005) and (c) a pre-motoneuronal spinal network of interneurons.

Previous investigators have evaluated the potential of non-uniform spatial distribution of mechanoreceptor input on the differential activation of the parasternal intercostal muscles in animal studies (De Troyer et al. 1996; Legrand et al. 1996), i.e. inspiratory activity is greatest and occurs earlier in the medial region of the parasternal muscles and occurs later and decreases progressively toward the costochrondral junction (De Troyer & Legrand, 1995; Legrand et al. 1996; DiMarco et al. 2005). In dog studies (De Troyer et al. 1996), following elimination of segmental afferent input to parasternal motoneurons by dorsal rhizotomy, the mediolateral gradient of parasternal activation persisted. Consequently, these investigators concluded that proprioceptor mechanisms did not play a significant role in the parasternal activation gradient. In contrast to the parasternals, however, the external intercostal muscles have a much higher density of muscle spindles (Duron et al. 1978; De Troyer et al. 2005). In addition, muscle spindle density manifests a rostrocaudal gradient (Duron et al. 1978). Notwithstanding the results concerning parasternal activation, therefore, the potential influence of proprioceptor influences on the spatial distribution of external intercostal activation should be explored in additional studies.

There is evidence for and against the possibility that differences in the intrinsic properties of the external intercostal motoneurons may play some role in the differential activation of the external intercostal muscles. Spontaneous breathing is characterized by an orderly recruitment of respiratory motor units which has been shown to follow Henneman's ‘size principle’ (Henneman et al. 1965; Berger, 1979; Dick et al. 1987b; Hilaire et al. 1987; Jodkowski et al. 1987; Sieck, 1988). Accordingly, smaller motoneurons have a lower threshold for activation than larger motoneurons because of their intrinsic electrophysiological properties (Iscoe et al. 1976; Enoka, 2002). More specifically, smaller motoneurons have a lower membrane surface area and higher input resistance and, therefore, are more excitable. Motor units with slower axonal conduction velocities and slow twitch oxidative fibres (Type I) correlate with this smaller motoneuron size and are recruited first during inspiration (Dick et al. 1987a; Sieck, 1988). Motoneuron activation and, therefore, motor unit recruitment proceeds in an orderly fashion from slow to fast units. Consistent with the concept that the intrinsic motoneuron properties play a role in the differential activation of the external intercostal muscles, previous investigators have shown that areas of the external intercostal muscles with high levels of drive, i.e. the dorsal portion of the rostral interspaces, had a higher proportion of Type I fibres compared to other areas of the external intercostal muscles in animal studies (Hardman & Brown, 1985; Greer & Martin, 1990). In the cat, Greer & Martin (1990) demonstrated that the external intercostal muscles in the dorsal portion of the rib cage were characterized by a rostrocaudal decrease in the proportion of Type I fibres. These motor units would be expected to have relatively smaller motoneurons and therefore be activated preferentially. Inconsistent with this hypothesis, however, Hardman & Brown (1985), in a rat model, found no difference in motoneuron size between distal and proximal external intercostal muscles. Of interest, other investigators also found no differences in motoneuron morphology among motoneurons innervating the medial and lateral portions of the parasternal muscles in dogs (Zhan et al. 2000). The potential role of differences in the intrinsic properties of the external intercostal motoneurons on the spatial distribution of external intercostal muscles, therefore, remains uncertain.

It is also possible that other motoneuron properties may mediate differential activation (De Troyer et al. 2005). One potential characteristic relates to the influence of persistent inward currents (PICs) mediated by Ca2+ channels (Collins et al. 2001; Hultborn et al. 2004). Motoneurons innervating different areas of the intercostal musculature may have differing amounts of these channels resulting in varying motoneuron output (De Troyer et al. 2005). Also, previous studies evaluating the differential activation of the parasternal intercostal muscles demonstrated that motoneurons innervating different portions of the parasternal muscles have differences in serotonergic inputs (Zhan et al. 2000). More specifically, the number of 5-hydroxytryptamine (5-HT) terminals and their density was greater in the motoneurons innervating the medial region of the parasternal muscle compared to the lateral region (Zhan et al. 2000). Interestingly, serotonin is also a known modulator of PICs. Finally, as suggested by Saboisky et al. (2007), a spinal pre-motoneuronal network of interneurons may also function to control differential activation of the external intercostal muscles. Supporting this thesis, a plethora of respiratory interneurons with inspiratory firing patterns is present in the lower cervical and upper thoracic spinal cord (Kirkwood et al. 1988; Palisses et al. 1989; Bellingham & Lipski, 1990).

Clinical implications

Based upon previous studies (DiMarco & Kowalski, 2009, 2010), inspiratory muscle activation via HF-SCS may offer a useful method to provide ventilatory support to patients with chronic respiratory failure secondary to cervical spinal cord injury. In fact, this technique has potential application in patients with and without phrenic nerve function, the latter of which are not candidates for conventional diaphragm pacing (DiMarco et al. 1994, 2005).

We have proposed in previous reports (DiMarco & Kowalski, 2009, 2010) that HF-SCS may provide a more physiological and potentially more effective method of inspiratory muscle activation. First, this method results in activation of the inspiratory muscles at firing frequencies in the physiological range (DiMarco & Kowalski, 2009). Second, by averting the need for chronic low frequency stimulation characteristic of conventional diaphragm pacing (Pette & Vrbová, 1999; DiMarco, 2005), fibre type conversion may be avoided resulting in preserved histological characteristics and mechanical function (DiMarco & Kowalski, 2010). Third, since this technique entails motoneuron stimulation, this method may provide an orderly pattern of motor unit recruitment (rather than synchronous activation of all motor units within the field of stimulation) resulting in a broader range of force modulation and consequently magnitude of inspired volume (Dick et al. 1987b; Mantilla & Sieck, 2003). Finally, this method results in synchronous activation of both the diaphragm and intercostal muscles which also enhances inspired volume generation (DiMarco et al. 1989; DiMarco & Kowalski, 2009).

It is important to note, however, that the stimulus train was applied in the shape of a square wave in the present investigation resulting in an altered pattern of inspiratory flow. Future studies involving a ramp application are necessary to achieve inspiratory flow patterns resembling spontaneous breathing. In addition, the rib cage muscle contribution to inspired volume was greater during HF-SCS compared to spontaneous breathing potentially resulting in a less efficient ventilatory pattern. Electrodes designed to provide more selective spinal cord pathway activation may allow more selective inspiratory muscle activation.

The fact that HF-SCS results in a distribution of inspiratory drive to the external intercostal muscles that resembles spontaneous breathing also has potential clinical significance. Importantly, several previous studies have demonstrated that the pattern of neural drive to the intercostal muscles matches the specific gradients of mechanical advantage of these muscles (Legrand & De Troyer, 1999; De Troyer et al. 2005). In addition, the topographical distribution of external intercostal muscle mass, in both dogs (De Troyer et al. 1999) and humans (De Troyer et al. 2005), is greatest in the dorsal half of the rostral interspaces and decreases both caudally and ventrally thus amplifying the respiratory effects of external intercostal muscle contraction in these areas of the rib cage. Teleologically and from a theoretical standpoint, therefore, it appears that this degree of matching would serve to minimize the metabolic cost of breathing (De Troyer et al. 2005). Finally, there is evidence that areas of intercostal muscles with greater neural drive have a higher proportion of fatigue-resistant muscle fibres (Greer & Martin, 1990). Given the highly repetitive nature of inspiratory muscle recruitment necessary to maintain ventilation, the observed spatial distribution of neural drive may also serve to minimize the potential for muscle fatigue. The results of this study, therefore, provide an additional compelling argument that HF-SCS results in a more physiological and potentially more effective method of inspiratory muscle activation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix

Appendix

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  8. Appendix

Author contributions

The experiments described in this manuscript were performed in the Department of Physiology and Biophysics, Case Western Reserve University at MetroHealth Medical Centre, Cleveland, OH, USA. A.D. and K.K. have both contributed to the conception and design of the experiments; the collection, analysis and interpretation of data; and drafting and revising the article critically for important intellectual content. Both authors approved the final version of the manuscript.

Acknowledgements

The authors wish to thank Ms Dana Hromyak for her technical expertise in the preparation of this manuscript. This work was supported by the National Institutes of Health: R01 NS064157.