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. Author manuscript; available in PMC: 2016 May 1.
Published in final edited form as: Respir Physiol Neurobiol. 2015 Jan 29;210:14–21. doi: 10.1016/j.resp.2015.01.013

Impact of Unilateral Denervation on Transdiaphragmatic Pressure

Luther C Gill 1, Carlos B Mantilla 1,2, Gary C Sieck 1,2
PMCID: PMC4449269  NIHMSID: NIHMS693277  PMID: 25641347

1. INTRODUCTION

Mechanical activation of the diaphragm muscle (DIAm) mediates airflow into the lungs by generating a pressure difference across the muscle—transdiaphragmatic pressure (Pdi). Previous work from our lab in cats (Fournier and Sieck, 1988b; Sieck and Fournier, 1989), hamsters (Sieck, 1994), rats (Mantilla et al., 2010) and more recently in mice (Greising et al., 2013) used Pdi measurements to estimate DIAm force generation across a range of ventilatory (rhythmic gas exchange) and higher force, non-ventilatory behaviors. In these studies we found that Pdi generated during ventilatory behaviors is consistently less than 50% of maximal Pdi (Pdimax) elicited by bilateral phrenic nerve stimulation. For example, Pdi generated during quiet rhythmic breathing (eupnea) ranged from 10–27% of Pdimax depending on species (Greising et al., 2013; Mantilla et al., 2010; Sieck and Fournier, 1989; Watchko et al., 1986). Stimulating breathing by exposure to a hypoxic-hypercapnic (10% O2–5% CO2) gas mixture increased Pdi generated during ventilatory behaviors; however, Pdi never exceeded 36% of Pdimax across several species (Greising et al., 2013; Mantilla et al., 2010; Sieck and Fournier, 1989; Watchko et al., 1986).

Although lung inflation may result, the goal of higher force, non-ventilatory behaviors of the DIAm is not gas exchange. Often DIAm activation during these behaviors is preparatory for expulsive airway clearance, e.g., coughing or sneezing. The Pdi generated during higher force, non-ventilatory behaviors is substantially greater than that generated during ventilatory behaviors (Greising et al., 2013; Mantilla et al., 2010; Sieck and Fournier, 1989; Watchko et al., 1986). For example in cats, mechanical stimulation of the oropharynx induces a gagging/coughing behavior in which Pdi approximates Pdimax (Sieck and Fournier, 1989). Also in cats, the Pdi generated during sneezing induced by mechanical stimulation of the nasopharynx was found to be maximal (comparable to Pdimax). Similarly in rats, a sneezing behavior induced by intranasal injection of capsaicin is associated with generation of near maximal Pdi (94% of Pdimax) (Mantilla et al., 2010). The Pdi’s generated during other higher force, non-ventilatory behaviors of the DIAm are also greater than during ventilatory behaviors. For example, during sustained airway occlusion, Pdi ranges from 43–70% of Pdimax across species (Greising et al., 2013; Mantilla et al., 2010; Sieck and Fournier, 1989; Watchko et al., 1986).

The DIAm comprises separate left and right hemidiaphragms, each with its own phrenic nerve innervation, with no crossover of innervation (Fournier and Sieck, 1988a). Accordingly, unilateral denervation (DNV) (Argadine et al., 2009; Geiger et al., 2001; Gosselin et al., 1994; Sieck, 1994; Sieck and Zhan, 2000) induces DIAm hemi-paralysis, reducing maximum force generating capacity of the DIAm by ~50%. We hypothesized that following unilateral DNV, the ability of the contralateral DIAm to generate sufficient Pdi to accomplish ventilatory behaviors will not be compromised and normal ventilation (as determined by arterial blood gas measurements) will not be impacted, although neural drive to the DIAm increases. In contrast, we hypothesized that those higher force, non-ventilatory behaviors requiring Pdi generation greater than 50% of Pdimax will be compromised following DIAm hemiparalysis, i.e., increased neural drive cannot compensate for lack of force generating capacity.

2. METHODS

All experiments were approved by the Institutional Animal Care and Use Committee of the Mayo Clinic. A total of 18 adult, male Sprague-Dawley rats (300–350 g) from Harlan Laboratories (Indianapolis, IN) were used for this study. Rats were anesthetized via intramuscular ketamine (90 mg/kg) and xylazine (10 mg/kg) injections for all experimental procedures. Animals were randomly assigned to either the sham control (n=6) or DNV (n=6) groups. Additional animals (n=6) were used to measure changes in ventilatory parameters and blood gases before and after DNV.

2.1. Denervation

The right phrenic nerve was isolated in the lower neck and sectioned. A 10–20 mm length of the nerve was removed to ensure complete DIAm DNV. In all animals, DIAm hemiparalysis was verified by the absence of EMG activity in the ipsilateral (right) DIAm. The sham group underwent a similar surgical procedure as the animals in the DNV group, but the right phrenic nerve remained intact.

2.2. Transdiaphragmatic Pressure Measurements

Measurements of Pdi were performed based on the difference between esophageal and gastric pressures as previously described (Fournier and Sieck, 1988a; Greising et al., 2013; Mantilla et al., 2010; Sassoon et al., 1996; Sieck and Fournier, 1989; Watchko et al., 1986). In anesthetized animals, two 3.5 French Millar solid-state pressure catheters (SPR-524; Millar Instruments, Houston, TX) were inserted through the mouth into the esophagus and stomach, spanning the thoracic and abdominal borders of the DIAm, respectively. Correct catheter position was determined based on the direction of signal deflection and postmortem analysis. Measurements were collected during the following conditions and sequence: (1) breathing of room air (eupnea) for 5 min, (2) exposure to a hypoxia (10% O2)–hypercapnia (5% CO2) gas mixture for 5 min, (3) sustained airway occlusion (by covering nose and mouth for ~40 s), (4) maximum Pdi (Pdimax) obtained by supramaximal bilateral phrenic nerve stimulation (using a stimulus isolation unit to control the current pulse) at 75 Hz (0.5 ms duration pulses in 300 ms trains repeated each s) using bipolar electrodes (FHC, Bowdoin, ME); and (5) sneezing, induced by intranasal infusion of 10 µl of 30 µM capsaicin. Measurements were also obtained when animals took deep breaths (“sighs”) defined as spontaneously occurring inspiratory events that were >2 times eupneic Pdi amplitude. Rats were given 5–10 min intervals between behaviors to allow for re-acclimatization to a tidal breathing condition and for Pdi amplitude to return to eupneic values.

Intra-thoracic and abdominal pressures were measured independently and recorded with a PowerLab 8/35 data acquisition system with an integrated amplifier following the manufacturer recommended calibration procedure. Pressure data were sampled at 100 Hz using LabChart (Millar Instrumentation) and band-pass filtered (0.3–30 Hz). Data from LabChart was exported to MATLAB for custom-designed automated analyses of peak pressure amplitudes and corresponding baselines on an event-by-event (e.g., breath-by-breath) basis. Baseline pressure values were determined systematically from the average of all inflection points in the segment preceding each peak. Data were analyzed and averaged across behaviors for 2 min of eupnea, 2 min of hypoxia–hypercapnia, and 5 maximal breaths during occlusion, all spontaneous deep breaths, all sneezes, and the maximal value obtained during bilateral phrenic nerve stimulation at 75 Hz. Pdi measurements across all conditions were obtained before and after unilateral DIAm DNV. Across all behaviors, movement of the abdomen was constrained using a custom-made binder to approximate isometric conditions (determined by Pdi response) and to minimize changes in functional residual capacity (FRC) of the lung.

2.3. Diaphragm Electromyography (EMG)

In all animals, DIAm EMG was measured using methods similar to those previously described (Dow et al., 2006; Mantilla et al., 2011; Mantilla et al., 2010; Sieck and Fournier, 1990; Trelease et al., 1982). Briefly, pairs of multistranded fine wire, insulated (stripped to expose ~2 mm segment) stainless steel electrodes (0.28 mm diameter – model AS631, Cooner Wire Inc., Chatsworth, CA) were implanted (~3 mm apart) into the mid-costal regions of both right and left sides of the DIAm following laparotomy. The compound EMG signal was differentially amplified (2000×), bandpass filtered (20–1000 Hz) using an analog amplifier (Model 2124, DATA Inc.) and digitally sampled at a frequency of 2 kHz using a data acquisition board (National Instruments, Austin, TX) controlled by a custom-made program (LabView 8.2; National Instruments). The root-mean squared (RMS) of the EMG signal was computed using a 100-ms window. Respiratory rate, inspiratory burst duration, duty cycle, the RMS value at 75 ms after the onset of DIAm activity (RMS75 – an estimate of neural drive (Seven et al., 2014), and peak RMS (RMSpeak) were determined from the EMG signal. The maximum DIAm RMS EMG value was estimated based on the positive linear relationship between DIAm RMS EMG and Pdi measurements that we previously observed (Mantilla et al., 2010). Accordingly, in each animal, the maximum DIAm RMS EMG value was extrapolated from RMS EMG and Pdi measurements obtained in other behaviors.

2.4. Plethysmography

A commercially available whole-body plethysmography system (Buxco Inc., Wilmington, NC, USA) was used to quantify ventilation in additional anesthetized animals. The plethysmograph was calibrated by injecting known volumes of gas into a Plexiglas recording chamber using a 5-mL syringe. The chamber pressure, temperature and humidity, and atmospheric pressure as well as the rectal temperature of the rat were used in an equation first described by (Drorbaugh and Fenn, 1955) to calculate respiratory volumes, and peak airflow rates before and after DNV. Ventilatory parameters were calculated from the airflow traces, which were continuously sampled at 500 Hz. During the experiments, gas mixtures flowed through the chamber at a rate of 2 L/min to enable control of inspired gases.

2.5. Arterial Blood Gas Measurements

Arterial PO2, PCO2 and pH (i-Stat, Heska, Fort Collins, CO) were measured during eupnea in a subset of animals (n=6) that did not undergo the sequence of ventilatory and non-ventilatory behaviors utilized during Pdi measurements. After animals were anesthetized, the right femoral artery was cannulated with a catheter (PE-10 tubing, OD 0.61 mm, ID 0.28mm) filled with heparinized saline. Arterial blood samples were drawn before (0.25 ml) and after (0.25 ml) unilateral DNV.

2.6. Statistical Analyses

Differences in Pdi across motor behaviors were evaluated using a two-way repeated measures ANOVA (condition: pre- and post-DNV × behavior) with Tukey–Kramer honestly significant difference post hoc tests when appropriate (Tukey, 1949). Differences in arterial blood gas values, respiratory rate, burst duration, duty cycle, DIAm RMSpeak and RMS75 EMG were determined using ANOVA and post hoc paired-sample t-tests. All statistical evaluations were performed using standard statistical software (JMP 8.0, SAS Institute Inc., Cary, NC). Statistical significance was established at the 0.05 level. All experimental data are presented as mean ± standard error (SE) across animals, unless otherwise specified.

3. RESULTS

In all animals, unilateral DNV and DIAm hemiparalysis was verified by the absence of EMG activity in the ipsilateral (right) DIAm (Fig. 1). Based on DIAm EMG recordings from the contralateral (left) DIAm during eupnea, respiratory rate increased from 63 ± 3 breaths/min to 85 ± 4 breaths/min (p< 0.0002) after unilateral DNV. Inspiratory burst duration also increased after DNV from 281 ± 3 ms to 349 ± 18 ms (p<0.005). The duty cycle (inspiratory burst duration divided by total respiratory cycle duration) of DIAm activity also increased from 30 ± 3% to 49 ± 4% after DNV (p<0.0001).

Figure 1.

Figure 1

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Representative measurements of transdiaphragmatic pressure (Pdi), raw diaphragm (DIAm) EMG (both left, L and right, R), and root mean squared (RMS) EMG before (PRE) and after (POST) unilateral (right, R) DNV in an adult male rat across 1) ventilatory behaviors: eupnea, hypoxia (10% O2)/hypercapnia (5% CO2) and spontaneous deep breaths; and 2) higher force, non-ventilatory behaviors: sustained airway occlusion and sneeze.

3.1. Transdiaphragmatic Pressure

Representative tracings of Pdi generated during ventilatory and higher force, non-ventilatory motor behaviors are shown in Figure 1. The maximum Pdi generated by bilateral phrenic nerve stimulation at 75 Hz was 36.6 ± 1.4 cm H2O. Figure 2 summarizes the impact of sham surgery and unilateral DNV on the Pdi’s generated during different ventilatory and higher force, non-ventilatory behaviors. There were no differences in Pdi’s generated during different ventilatory and higher force, non-ventilatory behaviors after sham surgery. After unilateral DNV, the Pdi generated by stimulation of the intact phrenic nerve was 23.1 ± 1.7 cm H2O (63% of Pdimax before DNV) (p<0.001). Before DNV, the average Pdi generated during eupnea was 8.6 ± 0.6 cm H2O (24% of Pdimax) and 9.8 ± 0.7 cm H2O (27% of Pdimax) during hypoxia–hypercapnia. After DNV, the Pdi generated during these ventilatory behaviors was unaffected; during eupnea, Pdi was 8.5 ± 0.6 cm H2O (23% of Pdimax) and 9.5 ± 0.5 cm H2O (26% of Pdimax) during hypoxia–hypercapnia. The Pdi’s generated by the DIAm during higher force, non-ventilatory behaviors were significantly reduced after DNV. The average Pdi generated during airway occlusion was reduced from 23.5 ± 1.6 cm H2O (64% of Pdimax) before DNV to 17.0 ± 0.9 cm H2O (49% of Pdimax) after DNV (p<0.01). The Pdi generated during deep breaths (sighs) was 19.6 ± 0.9 cm H2O (53% of Pdimax) before DNV to 16.8± 0.9 cm H2O (46% of Pdimax) after DNV (p<0.01). During sneezing, Pdi was 22.7 ± 0.6 cm H2O (62% of Pdimax) before DNV and this decreased to 15.6 ± 0.5 cm H2O (43% of Pdimax) after DNV (p<0.01).

Figure 2.

Figure 2

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Normalized Pdi (A and B) and RMS EMG (C and D) during ventilatory and higher force, non-ventilatory behaviors before (Pre, white bars) and after (Post, black bars) sham (A and C) or DNV (B and D) surgery. Note that compared to values before surgery, Pdi generated during higher force, non-ventilatory behaviors (airway occlusion, and sneezing) were significantly reduced after DNV. In addition, RMSpeak EMG increased significantly during eupnea and hypoxia-hypercapnia after DNV. * p<0.05, values are means ± SE.

3.2. Diaphragm Electromyography (EMG)

Representative traces for compound DIAm EMG and RMS EMG generated during ventilatory and higher force, non-ventilatory DIAm motor behaviors are shown in Figure 1. Figure 2 summarizes the impact of sham surgery and unilateral DNV on DIAm EMG RMSpeak (normalized to the estimated maximum RMS EMG - RMSmax) generated during different ventilatory and higher force, non-ventilatory behaviors. There were no differences in DIAm RMSpeak EMG generated during different ventilatory and higher force, non-ventilatory behaviors after sham surgery. Although the Pdi’s generated during ventilatory behaviors were unaffected by unilateral DNV, DIAm RMSpeak EMG increased significantly during these motor behaviors after DNV (p<0.01). In contrast, although the Pdi’s generated during higher force, non-ventilatory behaviors were reduced after unilateral DNV, DIAm RMSpeak EMG values during these motor behaviors were unaffected by DNV.

As previously observed (Mantilla et al., 2010), there was a strong correlation between Pdi and RMS EMG measures across motor behaviors (Fig. 3; r2=0.78; p<0.0001). After unilateral DNV, this correlation persisted but was shifted downward and to the right, i.e., greater DIAm RMSpeak EMG was observed for a given level of Pdi (p<0.01).

Figure 3.

Figure 3

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Relationship between DIAm EMG (normalized to RMSpeak) and Pdi (normalized to Pdimax) measurements in rats across different ventilatory and higher force, non-ventilatory behaviors before and after unilateral DNV

As previously described (Seven et al., 2014), the RMS75 EMG value was used to estimate neural drive to the contralateral DIAm. Representative traces for RMSpeak and RMS75 EMG generated during ventilatory and higher force, non-ventilatory DIAm motor behaviors are shown in Figure 4. In the control group, there were no differences in RMSpeak and RMS75 EMG during different ventilatory and higher force, non-ventilatory behaviors before and after sham surgery. In contrast, both RMSpeak and RMS75 EMG values increased significantly after DNV during ventilatory behaviors (Fig. 2 C&D; Fig. 4 A&B). During eupnea, RMS75 EMG increased from 11 ± 1% before DNV to 19 ± 2% after DNV. During hypoxia-hypercapnia, RMS75 EMG increased from 12 ± 1% before to 21 ± 2% after DNV. During airway occlusion, DIAm RMSpeak and RMS75 EMG values did not change after DNV (Fig. 2 C&D; Fig. 4 E&F). Before DNV, RMS75 EMG was 25 ± 3% during airway occlusion and 6 ± 2% after DNV.

Figure 4.

Figure 4

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Neural drive to the DIAm during different ventilatory and higher force, non-ventilatory behaviors was estimated by measuring the EMG RMS value at 75 ms after the onset of activity (RMS75). In addition, the peak EMG RMS value (RMSpeak) was measured. Both values were normalized to the estimated maximal RMS values (RMSmax) based on measures during airway occlusion. A) Averaged EMG RMS traces obtained during eupnea before surgery (intact) and after sham surgery or unilateral DNV. Dashed line at 75 ms indicates the point at which RMS75 was calculated. B) Normalized EMG RMS75 (white bars) and RMSpeak (black bars) values during eupnea increased significantly after DNV. C) Averaged EMG RMS traces during hypoxia-hypercapnia before (intact), and after sham surgery or unilateral DNV. D) Normalized EMG RMS75 (white bars) and RMSpeak (black bars) values during hypoxia-hypercapnia increased significantly after DNV. E) Averaged EMG RMS traces during airway occlusion before (intact) and after sham surgery or unilateral DNV. F) Normalized EMG RMS75 (white bars) and RMSpeak (black bars) values during airway occlusion did not change after DNV. * p<0.05, values are means ± SE.

3.3. Plethysmography and Arterial Blood Gases

Ventilatory parameters before and after DNV are shown in Table 1. Although these measures were obtained in a separate group of animals, respiratory rate and inspiratory time were comparable when measured using plethysmography to measurements obtained from the DIAm EMG. As a result of the increase in respiratory rate (by 48%; p<0.01) and despite a small decrease in tidal volume (17% reduction; p<0.01), minute ventilation increased by 24% (p<0.01) after DNV. However, this was not reflected by changes in blood gases (Table 2), suggesting a change in dead-space ventilation without a change in alveolar ventilation. Comparing before and after DNV, peak inspiratory flow during eupnea decreased by 12% (p<0.01), expiratory time decreased by 34% (p<0.001) after DNV.

Table 1.

Ventilatory parameters before (pre) and after (post) unilateral DNV.

Ventilatory parameter Pre-DNV Post-DNV
Respiratory rate (min−1) 61 ± 5 90 ± 5 *
Tidal volume (ml) 1.6 ± 0.1 1.3 ± 0.1 *
Ventilation (ml min−1) 90.1 ± 5.9 112.1 ± 10.0
Peak inspiratory flow (ml s−1) 9.0 ± 0.6 8.0 ± 0.6 *
Peak expiratory flow (ml s−1) 7.5 ± 0.4 7.4 ± 0.5
Inspiratory time (s) 0.28 ± 0.0 0.34 ± 0.0
Expiratory time (s) 0.7 ± 0.1 0.5 ± 0.0 *
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Values are means ± SE.

*

p<0.05

Table 2.

Arterial blood gas values before (pre) and after (post) unilateral DNV indicate that ventilation was maintained after DNV.

Blood gas value Pre-DNV Post-DNV
pH 7.41 ± 0.01 7.41 ± 0.01
PCO2 (mmHg) 39.7 ± 0.9 37.9 ± 0.8
PO2 (mmHg) 94.0 ± 1.5 92.4 ± 1.0
HCO3− (mM) 24.2 ± 0.5 23.4 ± 0.6
SaO2 (%) 98 ± 0 95 ± 0
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Values are means ± SE.

4. DISCUSSION

In the present study, we found that in rats, DIAm hemiparalysis induced by unilateral DNV did not affect the Pdi generated during ventilatory behaviors and did not affect normal ventilation (as determined by blood gas levels). However, neural drive to the contralateral DIAm (RMS75) increased significantly to compensate for the loss of force contributed by the right paralyzed DIAm. These results indicate that the force reserve of the contralateral DIAm is sufficient to sustain ventilation in the rat even after unilateral DIAm paralysis. In contrast, we found that higher force, non-ventilatory DIAm motor behaviors requiring greater than ~50% Pdimax were compromised after unilateral DIAm DNV. These results suggest that essential airway clearance behaviors (e.g., coughing, sneezing) may be affected when the force reserve of the whole DIAm is compromised.

There are a number of conditions that may induce unilateral hemidiaphragm paralysis including: pulmonary malignancy (Welvaart et al., 2011), postoperative thoracic surgery complications (Smith et al., 2013), indwelling central venous catheters (Takasaki and Arai, 2001), and high cervical spinal cord injury (Goshgarian, 2003; Gransee et al., 2013; Mantilla et al., 2013a; Mantilla et al., 2013b; Mantilla and Sieck, 2009; Miyata et al., 1995; Vinit et al., 2006; Zhan et al., 1997). In all these conditions, the substantial reserve capacity of the DIAm for generating force may be significantly compromised, even if ventilation appears unimpaired. Thus, Pdi assessments conducted across a range of ventilatory and non-ventilatory behaviors are necessary to fully evaluate the functional impact of respiratory and neuromuscular diseases that affect DIAm strength.

4.1. Changes in Neural Drive to the Contralateral DIAm and Ventilatory Patterns after DNV

After DNV, respiratory rate increased by 35–47% together with a 21–24% increase in inspiratory duration and a 63% increase in duty cycle. During eupnea, there was also a 12% decrease in PIF and 17% decrease in Vt. At the same time neural drive to the contralateral DIAm (RMS75) increased by 60%. This change in neural drive to the contralateral DIAm during eupnea is consistent with our previous reports (Mantilla et al., 2013b; Miyata et al., 1995; Prakash et al., 1999). In two separate rat models of DIAm hemiparalysis, unilateral DNV, and tetrodotoxin-induced phrenic nerve conduction blockade, we found that EMG RMSpeak of the intact, contralateral DIAm increased by ~50% during eupnea relative to measures before hemiparalysis (Miyata et al., 1995; Prakash et al., 1999). In the present study, EMG RMSpeak of the contralateral DIAm increased by 60% during eupnea after DNV. Such an increase in EMG RMSpeak might be expected since normally, the two sides of the DIAm act synergistically to generate Pdi. Accordingly, the increase in contralateral DIAm motor output after unilateral DNV is most likely the result of a compensatory increase in central drive to the phrenic motoneuron pool following synergist removal. The increase in RMS75 in the left DIAm found post-DNV supports such an increase in central drive (i.e., a compensatory loading effect). Unilateral DIAm DNV would amount to a substantial increase in load on the intact side of the DIAm. Consistent with such an increase in load, PIF decreased by 12%, while inspiratory duration increased after unilateral DNV. Thus, during eupnea it is likely that the velocity of shortening of the intact DIAm decreased in response to an increased load.

4.2. Motor Unit Recruitment after DNV

The changes in neural drive and breathing pattern after DIAm hemiparalysis are likely a compensatory strategy to maintain adequate ventilation using only the contralateral pool of DIAm motor units. As in skeletal muscles, motor units in the DIAm comprise motor neurons and the muscle fibers they innervate (Fournier and Sieck, 1988a; Sieck, 1994; Sieck and Fournier, 1989). The properties of both phrenic motor neurons and muscle fibers are matched (Enad et al., 1989; Sieck, 1994) and critically determine the efficacy of the central nervous system in accomplishing specific motor tasks. During neural activation, motor units are recruited in an orderly fashion, based on the intrinsic electrophysiological properties of motoneurons such that, for a given synaptic input, smaller more excitable motor neurons innervating more fatigue resistant muscle fibers (type I and IIa fibers comprising type S and type FR motor units) are recruited before larger motor neurons innervating more fatigable muscle fibers (type IIx and IIb fibers comprising type FInt and type FF motor units) (Butler et al., 1999; Henneman et al., 1965; Mantilla and Sieck, 2011; Sieck and Fournier, 1989). The recruitment order of motor units is not likely to change after acute DIAm hemiparalysis. Thus, after DNV, a larger fraction of the contralateral DIAm motor unit pool is required to accomplish ventilatory behaviors reflected by the marked increase in RMS75. However, the decreased total number of fatigue resistant motor units available after unilateral DNV may affect ventilatory behaviors as well. With a reduced total number of available fatigue resistant motor units and an insufficient number on the contralateral side, it may become necessary to recruit more fatigable motor units to perform ventilatory behaviors unless breathing strategy changes. After unilateral DNV, with a 50% decrease in the total number of DIAm motor units that can contribute to Pdi generation, it is not surprising that the ability of the DIAm to generate Pdi during higher force, non-ventilatory behaviors is compromised. What is surprising is that the Pdimax induced by phrenic nerve stimulation was reduced by only 37% after DNV.

In previous studies from our lab, a simple model was proposed for the recruitment of different motor unit types in the DIAm when accomplishing a range of ventilatory and non-ventilatory behaviors. In this model, eupnea is accomplished by recruitment of type S and type FR motor units across species (Fournier and Sieck, 1988a; Mantilla et al., 2010; Mantilla and Sieck, 2011; Sieck and Fournier, 1989). Typically, recruitment of more fatigable (FInt and FF) motor units in the rat DIAm is required only during higher force, non-ventilatory behaviors that are infrequent and shorter in duration. Following unilateral DNV, it is likely that an increase in central drive to the contralateral phrenic motor neuron pool is necessary to recruit additional motor units to maintain adequate ventilation. This may impinge on recruitment of more fatigable (FInt and FF) motor units, but this is difficult to assess.

4.3. Compensatory Respiratory Plasticity after DNV

Neural plasticity is an essential aspect of respiratory control, providing adaptive mechanisms at all levels (central, spinal, peripheral) in response to inadequate ventilation (Feldman et al., 2013; Mantilla and Sieck, 2003). Indeed, compensatory respiratory plasticity following unilateral DIAm hemiparalysis has been studied extensively for more than a century (Fuller et al., 2008; Golder et al., 2003; Golder et al., 2001; Goshgarian, 2003; Mantilla et al., 2013a; Mantilla et al., 2014a; Mantilla et al., 2013b; Miyata et al., 1995; Porter, 1895; Prakash et al., 1999; Sieck, 1994; Zhan et al., 1997). However, the precise mechanisms responsible for the regulation and integration of central respiratory drive have not yet been fully elucidated.

Unlike other neural systems involving precise control of motor behaviors, the neural circuitry controlling DIAm activation is unique because the phrenic motor neurons must be activated rhythmically and repeatedly to maintain ventilation. The coordination, and timing, of neural drive to the phrenic motoneuron pool is complex and integration may occur at various levels within the central nervous system. A compensatory increase in central respiratory drive or adaptations at the motor unit level all serve to preserve breathing capacity after perturbation (Butler, 2007; Johnson and Mitchell, 2013; Mantilla and Sieck, 2003, 2009; Miyata et al., 1995; Zhan et al., 1997).

A myriad of sensory afferent inputs have the potential to directly affect respiratory motor output, including detection of acute changes in arterial blood gases (O2, CO2 pH) via central (Richerson et al., 2005) and peripheral chemoreceptors (Kumar and Prabhakar, 2007), and changes in lung volume via pulmonary stretch receptors (Schelegle and Green, 2001). Collectively, these afferent signals may modulate the central pattern generator for respiration (Feldman et al., 2013; Mantilla et al., 2014b). In this respect, these compensatory mechanisms remained intact after unilateral DNV since ventilation was maintained with an increase in central drive to contralateral phrenic motor neuron pool.

The reduced PIF and rate of lung inflation was associated with a 19% decrease in Vt. Changes in lung volume may impact DIAm force generating capacity by altering the force-length relationship. Lung inflation beyond FRC causes a shortening of the DIAm and reduction in force development (De Troyer et al., 2005; Mier et al., 1990). However, it is unlikely that this occurred in the present study since the abdomen was bound to minimize changes in FRC. A comprehensive assessment of Pdi in response to stimulation of the phrenic nerves across a range of lung volumes above and below FRC before DNV was beyond the scope of the present study, but any change in FRC induced by DIAm hemiparalysis could certainly complicate Pdi generation. To avoid such complications, measurements were made with the abdomen bound to limit changes in FRC and to maintain isometric conditions across motor behaviors (Greising et al., 2013; Mantilla et al., 2010; Sieck and Fournier, 1989). In a previous study (Zhan et al., 1995), we directly examined changes in DIAm length (using sonomicrometry) induced by unilateral DNV. We found that there was no change in static resting length of the DIAm (e.g., as would be imposed by an increase in end-expiratory lung volume) and only very minimal changes in rhythmic passive length of the paralyzed DIAm imposed by continued activation of the intact contralateral side. These passive length changes depended on DIAm region with passive stretch in the costal region and passive lengthening in the sternal region due to differences in fiber orientation. After performing these in vivo measurements of DIAm length, the DIAm was excised and in vitro experiments were then conducted, in which it was demonstrated that the passive length changes of the paralyzed DIAm would have had minimal impact on the force generation.

After DNV, a compensatory loading effect on the contralateral DIAm may contribute to the observed increase in central drive to contralateral phrenic motor neurons. Typically, in skeletal muscle, muscle spindles (group Ia) and Golgi tendon organs (group Ib) respond to changes in muscle length and force, respectively. Thus, an increase in central respiratory drive after acute DNV may have been mediated by input from muscle spindles and/or Golgi tendon organs emanating from the DIAm or chest wall. Muscle spindles are rarely found in the DIAm with most if present, being found in the crural portion of the DIAm (Duron et al., 1978; Langford and Schmidt, 1983). The majority of proprioceptive afferents in the DIAm arise from Golgi tendon organs (Road, 1990) situated near the tendinous portion of the crural and ventral costal DIAm regions. It is possible that paralysis contralateral DIAm after DNV resulted in withdrawal of inhibitory input emanating from Gogi tendon organs on that side. Such an effect of Gogi tendon organ input has been previously reported as the phrenic-to-phrenic inhibitory reflex (De Troyer et al., 1999; De Troyer, 1998; Gill and Kuno, 1963).

5. Conclusions

Unilateral DIAm DNV and paralysis does not affect Pdi generation during ventilatory behaviors, while Pdi generation during higher force, non-ventilatory behaviors is significantly compromised. The maintenance of Pdi during ventilatory behaviors is the result of an increase in neural drive to the contralateral DIAm. These results are consistent with the presence of a large reserve capacity for ventilatory functions of the DIAm. There are a number of conditions that result in DIAm weakness, and the large reserve capacity of the DIAm for ventilatory functions may obscure the impact of weakness on the performance of important higher force, non-ventilatory functions during disease progression. Thus, it is important to fully evaluate DIAm motor performance in ventilatory and non-ventilatory motor behaviors.

Acknowledgements

This work was supported by NIH grants HL96750, and AG44615, and the Mayo Clinic. LCG was supported by an NIH T32 Training Grant HL105355.

REFERENCES

  1. Argadine HM, Hellyer NJ, Mantilla CB, Zhan WZ, Sieck GC. The effect of denervation on protein synthesis and degradation in adult rat diaphragm muscle. J Appl Physiol. 2009;107:438–444. doi: 10.1152/japplphysiol.91247.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Butler JE. Drive to the human respiratory muscles. Respir Physiol Neurobiol. 2007;159:115–126. doi: 10.1016/j.resp.2007.06.006. [DOI] [PubMed] [Google Scholar]
  3. Butler JE, McKenzie DK, Gandevia SC. Discharge properties and recruitment of human diaphragmatic motor units during voluntary inspiratory tasks. J Physiol. 1999;518(Pt 3):907–920. doi: 10.1111/j.1469-7793.1999.0907p.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. De Troyer A, Brunko E, Leduc D, Jammes Y. Reflex inhibition of canine inspiratory intercostals by diaphragmatic tension receptors. The Journal of physiology. 1999;514(Pt 1):255–263. doi: 10.1111/j.1469-7793.1999.255af.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. De Troyer A, Cappello M, Scillia P. Effect of inflation on the interaction between the left and right hemidiaphragms. J Appl Physiol. 2005;99:1301–1307. doi: 10.1152/japplphysiol.00192.2005. [DOI] [PubMed] [Google Scholar]
  6. De Troyer AD. The canine phrenic-to-intercostal reflex. The Journal of physiology. 1998;508(Pt 3):919–927. doi: 10.1111/j.1469-7793.1998.919bp.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dow DE, Mantilla CB, Zhan WZ, Sieck GC. EMG-based detection of inspiration in the rat diaphragm muscle. Conf Proc IEEE Eng Med Biol Soc. 2006;1:1204–1207. doi: 10.1109/IEMBS.2006.260688. [DOI] [PubMed] [Google Scholar]
  8. Drorbaugh JE, Fenn WO. A barometric method for measuring ventilation in newborn infants. Pediatrics. 1955;16:81–87. [PubMed] [Google Scholar]
  9. Duron B, Jung-Caillol MC, Marlot D. Myelinated nerve fiber supply and muscle spindles in the respiratory muscles of cat: quantitative study. Anatomy and embryology. 1978;152:171–192. doi: 10.1007/BF00315923. [DOI] [PubMed] [Google Scholar]
  10. Enad JG, Fournier M, Sieck GC. Oxidative capacity and capillary density of diaphragm motor units. J Appl Physiol. 1989;67:620–627. doi: 10.1152/jappl.1989.67.2.620. [DOI] [PubMed] [Google Scholar]
  11. Feldman JL, Del Negro CA, Gray PA. Understanding the rhythm of breathing: so near, yet so far. Annual Review of Physiology. 2013;75:423–452. doi: 10.1146/annurev-physiol-040510-130049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fournier M, Sieck GC. Mechanical properties of muscle units in the cat diaphragm. J Neurophysiol. 1988a;59:1055–1066. doi: 10.1152/jn.1988.59.3.1055. [DOI] [PubMed] [Google Scholar]
  13. Fournier M, Sieck GC. Somatotopy in the segmental innervation of the cat diaphragm. J Appl Physiol. 1988b;64:291–298. doi: 10.1152/jappl.1988.64.1.291. [DOI] [PubMed] [Google Scholar]
  14. Fuller DD, Doperalski NJ, Dougherty BJ, Sandhu MS, Bolser DC, Reier PJ. Modest spontaneous recovery of ventilation following chronic high cervical hemisection in rats. Exp Neurol. 2008;211:97–106. doi: 10.1016/j.expneurol.2008.01.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Geiger PC, Cody MJ, Macken RL, Bayrd ME, Sieck GC. Effect of unilateral denervation on maximum specific force in rat diaphragm muscle fibers. J Appl Physiol. 2001;90:1196–1204. doi: 10.1152/jappl.2001.90.4.1196. [DOI] [PubMed] [Google Scholar]
  16. Gill PK, Kuno M. Excitatory and Inhibitory Actions on Phrenic Motoneurones. The Journal of physiology. 1963;168:274–289. doi: 10.1113/jphysiol.1963.sp007192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Golder FJ, Fuller DD, Davenport PW, Johnson RD, Reier PJ, Bolser DC. Respiratory motor recovery after unilateral spinal cord injury: eliminating crossed phrenic activity decreases tidal volume and increases contralateral respiratory motor output. J Neurosci. 2003;23:2494–2501. doi: 10.1523/JNEUROSCI.23-06-02494.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Golder FJ, Reier PJ, Bolser DC. Altered respiratory motor drive after spinal cord injury: supraspinal and bilateral effects of a unilateral lesion. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2001;21:8680–8689. doi: 10.1523/JNEUROSCI.21-21-08680.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Goshgarian HG. Plasticity in Respiratory Motor Control: Invited Review: The crossed phrenic phenomenon: a model for plasticity in the respiratory pathways following spinal cord injury. J Appl Physiol. 2003;94:795–810. doi: 10.1152/japplphysiol.00847.2002. [DOI] [PubMed] [Google Scholar]
  20. Gosselin LE, Brice G, Carlson B, Prakash YS, Sieck GC. Changes in satellite cell mitotic activity during acute period of unilateral diaphragm denervation. J. Appl. Physiol. 1994;77:1128–1134. doi: 10.1152/jappl.1994.77.3.1128. [DOI] [PubMed] [Google Scholar]
  21. Gransee HM, Zhan WZ, Sieck GC, Mantilla CB. Targeted Delivery of TrkB Receptor to Phrenic Motoneurons Enhances Functional Recovery of Rhythmic Phrenic Activity after Cervical Spinal Hemisection. PloS one. 2013;8:e64755. doi: 10.1371/journal.pone.0064755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Greising SM, Sieck DC, Sieck GC, Mantilla CB. Novel method for transdiaphragmatic pressure measurements in mice. Respir Physiol Neurobiol. 2013;188:56–59. doi: 10.1016/j.resp.2013.04.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Henneman E, Somjen G, Carpenter DO. Functional significance of cell size in spinal motoneurons. J Neurophysiol. 1965;28:560–580. doi: 10.1152/jn.1965.28.3.560. [DOI] [PubMed] [Google Scholar]
  24. Johnson RA, Mitchell GS. Common mechanisms of compensatory respiratory plasticity in spinal neurological disorders. Respiratory physiology & neurobiology. 2013;189:419–428. doi: 10.1016/j.resp.2013.05.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kumar P, Prabhakar N. Sensing hypoxia: carotid body mechanisms and reflexes in health and disease. Respiratory physiology & neurobiology. 2007;157:1–3. doi: 10.1016/j.resp.2007.02.003. [DOI] [PubMed] [Google Scholar]
  26. Langford LA, Schmidt RF. An electron microscopic analysis of the left phrenic nerve in the rat. The Anatomical record. 1983;205:207–213. doi: 10.1002/ar.1092050211. [DOI] [PubMed] [Google Scholar]
  27. Mantilla CB, Gransee HM, Zhan WZ, Sieck GC. Motoneuron BDNF/TrkB signaling enhances functional recovery after cervical spinal cord injury. Exp Neurol. 2013a;247C:101–109. doi: 10.1016/j.expneurol.2013.04.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mantilla CB, Greising SM, Stowe JM, Zhan WZ, Sieck GC. TrkB Kinase Activity is Critical for Recovery of Respiratory Function after Cervical Spinal Cord Hemisection. Exp Neurol. 2014a;261:190–195. doi: 10.1016/j.expneurol.2014.05.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mantilla CB, Greising SM, Zhan WZ, Seven YB, Sieck GC. Prolonged C2 spinal hemisection-induced inactivity reduces diaphragm muscle specific force with modest, selective atrophy of type IIx and/or IIb fibers. Journal of applied physiology. 2013b;114:380–386. doi: 10.1152/japplphysiol.01122.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mantilla CB, Seven YB, Hurtado-Palomino JN, Zhan WZ, Sieck GC. Chronic assessment of diaphragm muscle EMG activity across motor behaviors. Respir Physiol Neurobiol. 2011;177:176–182. doi: 10.1016/j.resp.2011.03.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mantilla CB, Seven YB, Sieck GC. Convergence of pattern generator outputs on a common mechanism of diaphragm motor unit recruitment. Progress in brain research. 2014b;209:309–329. doi: 10.1016/B978-0-444-63274-6.00016-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mantilla CB, Seven YB, Zhan WZ, Sieck GC. Diaphragm motor unit recruitment in rats. Respir Physiol Neurobiol. 2010;173:101–106. doi: 10.1016/j.resp.2010.07.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mantilla CB, Sieck GC. Mechanisms underlying motor unit plasticity in the respiratory system. J Appl Physiol. 2003;94:1230–1241. doi: 10.1152/japplphysiol.01120.2002. [DOI] [PubMed] [Google Scholar]
  34. Mantilla CB, Sieck GC. Neuromuscular adaptations to respiratory muscle inactivity. Respir Physiol Neurobiol. 2009;169:133–140. doi: 10.1016/j.resp.2009.09.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mantilla CB, Sieck GC. Phrenic motor unit recruitment during ventilatory and non-ventilatory behaviors. Respir Physiol Neurobiol. 2011;179:57–63. doi: 10.1016/j.resp.2011.06.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mier A, Brophy C, Moxham J, Green M. Influence of lung volume and rib cage configuration on transdiaphragmatic pressure during phrenic nerve stimulation in man. Respiration physiology. 1990;80:193–202. doi: 10.1016/0034-5687(90)90083-b. [DOI] [PubMed] [Google Scholar]
  37. Miyata H, Zhan WZ, Prakash YS, Sieck GC. Myoneural interactions affect diaphragm muscle adaptations to inactivity. J Appl Physiol. 1995;79:1640–1649. doi: 10.1152/jappl.1995.79.5.1640. [DOI] [PubMed] [Google Scholar]
  38. Porter WT. The Path of the Respiratory Impulse from the Bulb to the Phrenic Nuclei. J Physiol. 1895;17:455–485. doi: 10.1113/jphysiol.1895.sp000553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Prakash YS, Miyata H, Zhan WZ, Sieck GC. Inactivity-induced remodeling of neuromuscular junctions in rat diaphragmatic muscle. Muscle Nerve. 1999;22:307–319. doi: 10.1002/(sici)1097-4598(199903)22:3<307::aid-mus3>3.0.co;2-m. [DOI] [PubMed] [Google Scholar]
  40. Richerson GB, Wang W, Hodges MR, Dohle CI, Diez-Sampedro A. Homing in on the specific phenotype(s) of central respiratory chemoreceptors. Experimental physiology. 2005;90:259–266. doi: 10.1113/expphysiol.2005.029843. discussion 266-259. [DOI] [PubMed] [Google Scholar]
  41. Road JD. Phrenic afferents and ventilatory control. Lung. 1990;168:137–149. doi: 10.1007/BF02719685. [DOI] [PubMed] [Google Scholar]
  42. Sassoon CS, Gruer SE, Sieck GC. Temporal relationships of ventilatory failure, pump failure, and diaphragm fatigue. J Appl Physiol. 1996;81:238–245. doi: 10.1152/jappl.1996.81.1.238. [DOI] [PubMed] [Google Scholar]
  43. Schelegle ES, Green JF. An overview of the anatomy and physiology of slowly adapting pulmonary stretch receptors. Respiration physiology. 2001;125:17–31. doi: 10.1016/s0034-5687(00)00202-4. [DOI] [PubMed] [Google Scholar]
  44. Seven YB, Mantilla CB, Sieck GC. Recruitment of Rat Diaphragm Motor Units Across Motor Behaviors with Different Levels of Diaphragm Activation. J Appl Physiol, jap.01395.02013. 2014 doi: 10.1152/japplphysiol.01395.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sieck GC. Physiological effects of diaphragm muscle denervation and disuse. Clin Chest Med. 1994;15:641–659. [PubMed] [Google Scholar]
  46. Sieck GC, Fournier M. Diaphragm motor unit recruitment during ventilatory and nonventilatory behaviors. J Appl Physiol. 1989;66:2539–2545. doi: 10.1152/jappl.1989.66.6.2539. [DOI] [PubMed] [Google Scholar]
  47. Sieck GC, Fournier M. Changes in diaphragm motor unit EMG during fatigue. J Appl Physiol. 1990;68:1917–1926. doi: 10.1152/jappl.1990.68.5.1917. [DOI] [PubMed] [Google Scholar]
  48. Sieck GC, Zhan WZ. Denervation alters myosin heavy chain expression and contractility of developing rat diaphragm muscle. J. Appl. Physiol. 2000;89:1106–1113. doi: 10.1152/jappl.2000.89.3.1106. [DOI] [PubMed] [Google Scholar]
  49. Smith BM, Ezeokoli NJ, Kipps AK, Azakie A, Meadows JJ. Course, predictors of diaphragm recovery after phrenic nerve injury during pediatric cardiac surgery. The Annals of thoracic surgery. 2013;96:938–942. doi: 10.1016/j.athoracsur.2013.05.057. [DOI] [PubMed] [Google Scholar]
  50. Takasaki Y, Arai T. Transient right phrenic nerve palsy associated with central venous catheterization. British journal of anaesthesia. 2001;87:510–511. doi: 10.1093/bja/87.3.510. [DOI] [PubMed] [Google Scholar]
  51. Trelease RB, Sieck GC, Harper RM. A new technique for acute and chronic recording of crural diaphragm EMG in cats. Electroencephalogr Clin Neurophysiol. 1982;53:459–462. doi: 10.1016/0013-4694(82)90011-6. [DOI] [PubMed] [Google Scholar]
  52. Tukey JW. Comparing individual means in the analysis of variance. Biometrics. 1949;5:99–114. [PubMed] [Google Scholar]
  53. Vinit S, Gauthier P, Stamegna JC, Kastner A. High cervical lateral spinal cord injury results in long-term ipsilateral hemidiaphragm paralysis. J Neurotrauma. 2006;23:1137–1146. doi: 10.1089/neu.2006.23.1137. [DOI] [PubMed] [Google Scholar]
  54. Watchko JF, Mayock DE, Standaert A, Woodrum DE. Postnatal changes in transdiaphragmatic pressure in piglets. Pediatr Res. 1986;20:658–661. doi: 10.1203/00006450-198607000-00016. [DOI] [PubMed] [Google Scholar]
  55. Welvaart WN, Paul MA, van Hees HW, Stienen GJ, Niessen JW, de Man FS, Sieck GC, Vonk-Noordegraaf A, Ottenheijm CA. Diaphragm muscle fiber function and structure in humans with hemidiaphragm paralysis. Am J Physiol Lung Cell Mol Physiol. 2011;301:L228–L235. doi: 10.1152/ajplung.00040.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Zhan WZ, Farkas GA, Schroeder MA, Gosselin LE, Sieck GC. Regional adaptations of rabbit diaphragm muscle fibers to unilateral denervation. J Appl Physiol. 1995;79:941–950. doi: 10.1152/jappl.1995.79.3.941. (1985) [DOI] [PubMed] [Google Scholar]
  57. Zhan WZ, Miyata H, Prakash YS, Sieck GC. Metabolic and phenotypic adaptations of diaphragm muscle fibers with inactivation. Journal of applied physiology. 1997;82:1145–1153. doi: 10.1152/jappl.1997.82.4.1145. [DOI] [PubMed] [Google Scholar]

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