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. 2016 Nov 9;117(2):537–544. doi: 10.1152/jn.00654.2016

BDNF effects on functional recovery across motor behaviors after cervical spinal cord injury

Vivian Hernandez-Torres 1,2, Heather M Gransee 1,2, Carlos B Mantilla 1,2, Yao Wang 2, Wen-Zhi Zhan 1, Gary C Sieck 1,2,✉
PMCID: PMC5288474  PMID: 27832605

This study demonstrates that after unilateral C2 spinal cord hemisection (SH), there are differences in the spontaneous recovery of diaphragm (DIAm) electromyographic activity during ventilatory compared with more forceful, nonventilatory motor behaviors. Furthermore, we show that intrathecal delivery of brain-derived neurotrophic factor (BDNF) at the level of the phrenic motor neuron pool enhances recovery of ipsilateral DIAm activity following SH, exerting main effects on recovery of ventilatory but not higher force, nonventilatory behaviors.

Keywords: neurotrophin, spinal hemisection, motor unit recruitment, respiration, respiratory muscle, spinal cord injury

Abstract

Unilateral C2 cervical spinal cord hemisection (SH) disrupts descending excitatory drive to phrenic motor neurons, thereby paralyzing the ipsilateral diaphragm muscle (DIAm) during ventilatory behaviors. Recovery of rhythmic DIAm activity ipsilateral to injury occurs over time, consistent with neuroplasticity and strengthening of spared synaptic inputs to phrenic motor neurons. Localized intrathecal delivery of brain-derived neurotrophic factor (BDNF) to phrenic motor neurons after SH enhances recovery of eupneic DIAm activity. However, the impact of SH and BDNF treatment on the full range of DIAm motor behaviors has not been fully characterized. We hypothesized that all DIAm motor behaviors are affected by SH and that intrathecal BDNF enhances the recovery of both ventilatory and higher force, nonventilatory motor behaviors. An intrathecal catheter was placed in adult, male Sprague-Dawley rats at C4 to chronically infuse artificial cerebrospinal fluid (aCSF) or BDNF. DIAm electromyography (EMG) electrodes were implanted bilaterally to record activity across motor behaviors, i.e., eupnea, hypoxia-hypercapnia (10% O2 and 5% CO2), sighs, airway occlusion, and sneezing. After SH, ipsilateral DIAm EMG activity was evident in only 43% of aCSF-treated rats during eupnea, and activity was restored in all rats after BDNF treatment. The amplitude of DIAm EMG (root mean square, RMS) was reduced following SH during eupnea and hypoxia-hypercapnia in aCSF-treated rats, and BDNF treatment promoted recovery in both conditions. The amplitude of DIAm RMS EMG during sighs, airway occlusion, and sneezing was not affected by SH or BDNF treatment. We conclude that the effects of SH and BDNF treatment on DIAm activity depend on motor behavior.

NEW & NOTEWORTHY This study demonstrates that after unilateral C2 spinal cord hemisection (SH), there are differences in the spontaneous recovery of diaphragm (DIAm) electromyographic activity during ventilatory compared with more forceful, nonventilatory motor behaviors. Furthermore, we show that intrathecal delivery of brain-derived neurotrophic factor (BDNF) at the level of the phrenic motor neuron pool enhances recovery of ipsilateral DIAm activity following SH, exerting main effects on recovery of ventilatory but not higher force, nonventilatory behaviors.

brain-derived neurotrophic factor (BDNF) signaling through its high-affinity tropomyosin-related kinase subtype B receptor (TrkB) plays an essential role in neuroplasticity following spinal cord injury (Bregman et al. 2002; Coumans et al. 2001; Kang and Schuman 1995; Mantilla et al. 2013a; Weishaupt et al. 2012), contributing to spontaneous recovery over time (Gill et al. 2016; Gransee et al. 2013, 2015; Mantilla et al. 2013a; Martinez-Galvez et al. 2016). For example, intrathecal delivery of BDNF promotes functional recovery of diaphragm muscle (DIAm) activity following unilateral spinal cord hemisection at the C2 cervical level (SH) (Bregman et al. 1997; Mantilla et al. 2013a; Novikova et al. 2000, 2002; Ye and Houle 1997). Similarly, transplantation of bone marrow-derived stem cells engineered to release BDNF enhances functional recovery of DIAm activity (Gransee et al. 2015; Lu et al. 2005). Postsynaptically, BDNF/TrkB signaling influences glutamate receptor activity by inducing phosphorylation of the NMDA receptor (Gottschalk et al. 1999; Lessmann et al. 1994), and such an effect could mediate neuroplasticity following SH. There is evidence for a role of BDNF/TrkB signaling in promoting recovery of rhythmic DIAm activity following SH via an upregulation of NMDA receptor and serotonergic receptor (5-HT2A) expression in phrenic motor neurons (Hadley et al. 1999; Mantilla et al. 2012; Zhou and Goshgarian 2000).

The DIAm is the primary inspiratory muscle and is innervated by phrenic motor neurons in the C3–C5 levels of the spinal cord in rats. Motor units are the final common pathway of neuromotor control, and recruitment of DIAm motor units plays a primary role in sustaining ventilatory behaviors (e.g., resting breathing/eupnea, exposure to hypoxia-hypercapnia conditions) as well as during high-force, nonventilatory behaviors related to airway clearance (e.g., airway occlusion, coughing, and sneezing) (Mantilla et al. 2010, 2014b; Seven et al. 2014; Sieck and Fournier 1989). Following SH, ipsilateral descending excitatory motor drive to phrenic motor neurons emanating from the medulla is disrupted, resulting in paralysis of the ipsilateral DIAm (Goshgarian et al. 1991; Mantilla et al. 2007, 2012, 2013b; Miyata et al. 1995; Prakash et al. 1995; Sieck 1994; Zhan et al. 1997). Contralateral descending medullary drive to phrenic motor neurons is spared and can be strengthened over time, resulting in recovery of rhythmic DIAm activity, although eupneic activity remains reduced compared with that before SH (Gransee et al. 2013, 2015; Mantilla et al. 2013a, 2013b; Martinez-Galvez et al. 2016). The impact of SH on descending inspiratory-related synaptic input to phrenic motor neurons is well established, but there is little information regarding the impact of SH on higher force, nonventilatory behaviors. Indeed, it is possible that the extent of unilateral vs. bilateral input may vary across motor behaviors, thus affecting the impact of SH.

If the effect of BDNF/TrkB signaling is primarily postsynaptic, and the extent of unilateral versus bilateral input to phrenic motor neurons is the same, then there should be a proportionate strengthening of the recovery of DIAm activity across all ventilatory and nonventilatory motor behaviors. Accordingly, we hypothesized that all DIAm motor behaviors are affected by SH and that intrathecal BDNF enhances the recovery of both ventilatory and nonventilatory motor behaviors after SH.

MATERIALS AND METHODS

Experimental animals.

A total of 16 Sprague-Dawley adult male rats (Harlan, Indianapolis, IN) with initial body weight of 280–300 g successfully completed this study. All experimental procedures were approved by the Institutional Animal Care and Use Committee at Mayo Clinic. Rats were anesthetized with an intramuscular injection of ketamine (90 mg/kg) and xylazine (10 mg/kg) for all surgical procedures and experimental measurements. All rats were subjected to a right-sided spinal cord hemisection at C2 (SH) and were randomly assigned to either an artificial cerebrospinal fluid (aCSF; n = 7)- or intrathecal BDNF (n = 8)-treated group.

Chronic DIAm EMG activity.

The method for DIAm EMG recording has been previously described in detail (Dow et al. 2006, 2009; Gransee et al. 2013, 2015; Mantilla et al. 2011, 2013a, 2013b; Martinez-Galvez et al. 2016; Sieck and Fournier 1990; Trelease et al. 1982). Briefly, 3 days before the SH surgery, animals were anesthetized and, following a midline laparotomy, two pairs of insulated fine wire electrodes (AS631; Cooner Wire, Chatsworth, CA) were implanted into the mid-costal region of the DIAm with an inter-electrode distance of ∼3 mm. The wires were subcutaneously tunneled to the dorsum of the animal and connected to an externalized nano circular connector (A79102-001/A79103-001; Omnetics Connector, Minneapolis, MN) at the dorsal surface between the scapulae for chronic EMG recordings. The surgical wounds were closed using 4-0 Vicryl sutures, and animals were allowed to recover for 3 days.

Spinal cord hemisection.

The surgical procedure for SH was previously described in detail (Gransee et al. 2013, 2015; Mantilla et al. 2013a, 2013b, 2014a; Martinez-Galvez et al. 2016; Miyata et al. 1995; Prakash et al. 1999; Zhan et al. 1997). Briefly, using a sterile technique, a dorsal laminectomy was performed at the C2 level, and the right anterolateral cord was transected using a microknife. Thereafter, muscle and skin layers were sutured (4-0 Vicryl sutures), and rats were allowed to recover on a heating pad before being returned to the animal area. All animals were observed daily after surgery and administered intramuscular buprenorphine (0.1 mg/kg) for the first 3 days following surgery and oral acetaminophen (100–300 mg/kg). Completeness of SH was verified by absence of ipsilateral eupneic DIAm EMG activity during surgery and 3 days post-SH (SH 3D).

Intrathecal BDNF infusion.

The method for intrathecal infusion at the level of the cervical spinal cord has been previously described (Mantilla et al. 2013a). At the time of SH surgery, an intrathecal catheter (PE-10; inner diameter 0.14 mm, outer diameter 0.4 mm; Becton Dickinson, Franklin Lakes, NJ) was inserted into the cisternal membrane and advanced 10 mm beyond the occipital crest such that the tip was placed at the C4 level of the spinal cord. The catheter was secured to the skull, and a connecting segment of PE-50 tubing was used to provide tension relief by leaving a loose loop of catheter in the neck. To deliver intrathecal BDNF (R&D Systems, Minneapolis, MN) or aCSF for 14 days, a mini-osmotic pump (Alzet 2002; Cupertino, CA) was implanted in the animal's dorsum. The infusion rate was 12 μl/day, with continuous BDNF delivery of 180 ng/day. The BDNF infusion was controlled to start at SH 3D by filling the distal end of the catheter with aCSF (10-cm length given the infusion flow rate of the mini-osmotic pump). The absence of ipsilateral DIAm EMG activity during eupnea was verified at SH 3D before intrathecal BDNF or aCSF treatment was initiated.

Chronic DIAm EMG recordings across motor behaviors.

The chronic monitoring of DIAm EMG activity has been previously described in detail (Mantilla et al. 2010, 2011). Immediately before SH surgery (pre-SH) and at 14 days post-SH (SH 14D), DIAm EMG activity was recorded in anesthetized animals during four different motor behaviors: 1) eupnea for ∼2 min, 2) hypoxia (10% O2)-hypercapnia (5% CO2) for 5 min, 3) airway occlusion induced by forced closure of the airway for 45 s, and 4) sneezing induced by intranasal capsaicin (10 μl, 30 mM; Cayman Chemical, Ann Arbor, MI). There was a 5-min rest period between each behavior to allow restoration of a normal breathing pattern. Eupneic DIAm EMG activity was also recorded in anesthetized rats during SH surgery, at SH 3D to verify completeness of SH by the absence of ipsilateral DIAm activity, and at SH 7D.

In all cases, data collection was conducted using LabView software (National Instruments, Austin, TX) at a sampling frequency of 2 kHz, followed by filtering (bandpass, 20-1,000 Hz) and amplification (×2,000). Root mean square (RMS) DIAm EMG activity was calculated using a 50-ms window (Mantilla et al. 2010, 2011; Seven et al. 2013; Sieck and Fournier 1990). EMG signals were analyzed using MATLAB 8.2 (The MathWorks, Natick, MA). The mean peak RMS EMG activity was measured during eupnea (2-min period), hypoxia-hypercapnia (exposed for a 5-min period and recording analyzed during the last 1 min), airway occlusion (occlusions held for 45 s and recordings analyzed in the last 5 s), and sneezing (typically multiple sneezing events with the recordings reflecting the highest amplitude event). In addition, spontaneous deep breaths (“sighs” defined as inspiratory events with amplitude at least twice eupneic amplitude) were recorded during periods of eupnea or hypoxia-hypercapnia. As a control for the quality of the EMG recordings, exclusion criteria based on amplitude of RMS EMG during sighs were established a priori on the basis of previous experiments measuring DIAm EMG activity across motor behaviors (Mantilla et al. 2011). Animals were included in the study only if the pre-SH DIAm RMS EMG sigh amplitude was 2.0–3.5 times the pre-SH eupneic amplitude.

Evidence for functional recovery included the presence of DIAm EMG activity that 1) was in phase with the contralateral side, 2) comprised more than one motor unit, and 3) had eupneic amplitude >10% of the pre-SH eupneic amplitude. Respiratory rate and duty cycle were calculated from contralateral DIAm EMG recordings pre-SH and at SH 14D.

Statistical analyses.

Statistical evaluations were performed using JMP statistical software (JMP 10.0; SAS Institute, Cary, NC). The sample size was based on a priori power analysis of DIAm RMS EMG activity in control animals across the range of motor behaviors. A statistical power analysis was performed for sample size estimation, based on data from previous publications of our laboratory (Gransee et al. 2013; Mantilla et al. 2013a). The effect size in this study was 0.05, with an alpha (α) value of 0.05 and a power of 0.8. The projected sample size needed with this effect size is eight animals per group. Thus our proposed sample is more than adequate for the main objective of this study. The proportion of animals displaying functional recovery of rhythmic inspiratory-related DIAm EMG activity was compared across groups using Pearson's χ2 test. DIAm RMS EMG amplitude was normalized to the pre-SH sigh value for each animal, and differences between treatment groups were examined using two-way analysis of variance (ANOVA; pre-SH vs. 14-day post-SH and treatment group) for each behavior. Respiratory rate and duty cycles were compared using repeated-measures ANOVA (pre- vs. 14-day post-SH and treatment group). Extreme values were considered outliers if the difference from the 25th and 75th percentile exceeded 1.5 times the interquartile range and were removed from analyses of EMG amplitude. In the BDNF group, one animal had outlier values bilaterally for sigh and occlusion and contralaterally for sneeze at SH 14D and one animal ipsilaterally for occlusion pre-SH. These animals were excluded from these analyses. When applicable, post hoc analyses were conducted using Tukey-Kramer's honestly significant difference test. Data are means ± SE unless otherwise specified. Statistical significance was established at P < 0.05.

RESULTS

Electrode implantation and surgical outcomes after SH.

Bilateral DIAm EMG electrodes were implanted at 3 days before SH for chronic monitoring of DIAm EMG activity for a period of up to 14 days post-SH. As in previous studies, DIAm EMG recordings were used during the SH surgery and at SH 3D to verify complete interruption of ipsilateral descending rhythmic excitatory premotor drive to phrenic motor neurons (Gransee et al. 2013, 2015; Mantilla et al. 2013a, 2013b). Despite loss of ipsilateral inspiratory-related DIAm EMG activity immediately after SH, all animals maintained normal spontaneous ventilation and none required mechanical ventilatory support. Mini-osmotic pumps were implanted for intrathecal delivery of aCSF or BDNF throughout the 14 days of the experiment. The intrathecal catheter tip location was verified visually at the terminal experiment. In a previous study (Mantilla et al. 2013a), we found that the intrathecal infusion rate (12 μl/day) localized BDNF delivery to cervical spinal cord segments corresponding to the phrenic motor nucleus in rats. Based on the intrathecal infusion rate that was used, it is likely that BDNF delivery was localized to the segments surrounding C4 corresponding to the phrenic motor pool in rats (Mantilla et al. 2009, 2013a). This assumption is consistent with the limited staining by bromophenol blue dye when injected intrathecally as repeated bolus injections up to 10 μl (Yaksh et al. 1997; Yaksh and Rudy 1976).

Proportion of animals displaying recovery of ipsilateral inspiratory-related DIAm EMG activity after SH.

Over time post-SH, spontaneous recovery of ipsilateral inspiratory-related DIAm EMG activity was evident in a subset of SH animals. Representative DIAm EMG recordings and RMS EMG measurements are shown in Fig. 1. Contralateral and ipsilateral inspiratory-related DIAm EMG activity occurred in rhythmic bursts in all lightly anesthetized animals. Intrathecal BDNF treatment increased the proportion of animals displaying recovery of DIAm EMG activity during eupnea after SH. At SH 7D, six of eight (75%) BDNF-treated SH animals displayed recovery of inspiratory-related DIAm EMG activity compared with 2 of 7 (29%) aCSF-treated SH animals (P = 0.072). At SH 14D, all BDNF-treated SH animals displayed recovery of ipsilateral DIAm EMG activity during eupnea compared with 3 of 7 (43%) aCSF-treated SH animals (P = 0.013). All SH animals, regardless of aCSF or BDNF treatment, displayed inspiratory-related DIAm EMG activity during hypoxia-hypercapnia and sighs. Similarly, all animals, regardless of treatment group, displayed ipsilateral DIAm EMG activity during airway occlusion and sneezing behaviors at SH 14D (see Figs. 1–3).

Fig. 1.

Fig. 1.

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Representative raw and root mean square (RMS) diaphragm (DIAm) EMG tracings from an animal in each treatment group (intrathecal aCSF and BDNF) obtained before and after SH across the following ventilatory and nonventilatory motor behaviors: eupnea, hypoxia-hypercapnia (10% O2-5% CO2), sigh, airway occlusion, and sneezing induced by intranasal capsaicin administration. All animals were chronically monitored using EMG recordings obtained by implanting DIAm electrodes. EMG was recorded before (pre-SH) and 14 days after SH (SH 14D). Notice that DIAm EMG activity occurs in bursts, reflecting rhythmic inspiration in anesthetized animals. DIAm EMG activity and RMS EMG amplitude increase significantly from ventilatory behaviors to nonventilatory behaviors before SH, consistent with previous results (Mantilla et al. 2010, 2011). DIAm EMG tracings represent the ipsilateral activity recorded from an animal in each of the SH 14D + aCSF and SH 14D + BDNF groups. Absence of ipsilateral DIAm EMG activity was verified in all animals at the time of SH surgery and at SH 3D (see materials and methods).

Fig. 3.

Fig. 3.

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Individual animal changes in DIAm RMS EMG amplitude post-SH with aCSF or BDNF treatment. DIAm RMS EMG amplitude (normalized to pre-SH sigh value for the same animal) is shown for each animal before (Pre) and 14 days after SH (SH) with aCSF and BDNF treatment. A: changes in the amplitude of the contralateral DIAm RMS EMG activity across motor behaviors and treatment conditions varied widely across animals. B: changes in ipsilateral DIAm RMS EMG activity were apparent across animals, with more animals displaying increasing RMS EMG activity after SH with BDNF compared with aCSF treatment during eupnea and hypoxia-hypercapnia, but not other motor behaviors.

Extent of recovery of ipsilateral DIAm EMG activity following SH.

Amplitude of DIAm RMS EMG activity was measured at SH 14D to assess the extent of recovery. The amplitude of DIAm RMS EMG at SH 14D was normalized to the sigh value measured pre-SH for the same animal. Figure 2 shows the summarized results across all animals, whereas Fig. 3 shows results for each animal at pre-SH and SH 14D. There were no differences between the aCSF- and BDNF-treated animals in the pre-SH values for any motor behavior, and pooled results are shown in Fig. 2.

Fig. 2.

Fig. 2.

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Extent of contralateral and ipsilateral rhythmic DIAm EMG activity at 14 days post-SH (SH 14D) following intrathecal aCSF or BDNF treatment. DIAm RMS EMG amplitude was measured during different motor behaviors at SH 14D and compared with the pre-SH RMS EMG amplitude. The amplitude of DIAm RMS EMG during each behavior was normalized to the sigh value measured pre-SH for the same animal. A: the normalized DIAm RMS EMG activity contralateral to SH was consistent in both groups at SH 14D compared with pre-SH, except for the hypoxia-hypercapnia (HH) condition, during which EMG amplitude was significantly greater after BDNF treatment compared with pre-SH (*P < 0.05). No difference was evident between the aCSF and BDNF treatment groups for any behavior. B: the normalized DIAm RMS EMG activity ipsilateral to SH was reduced in both the aCSF- and BDNF-treated groups compared with pre-SH during eupnea. Compared with aCSF treatment, intrathecal treatment with BDNF significantly increased ipsilateral DIAm EMG activity during eupnea and hypoxia-hypercapnia after SH (†P < 0.05). During sigh, occlusion, or sneezing, DIAm EMG activity was not different from that pre-SH in either the aCSF or BDNF treatment groups.

During eupnea, there was a statistically significant SH-induced decrease in inspiratory-related DIAm RMS EMG amplitude that was affected by BDNF treatment (2-way ANOVA; overall: P < 0.001; pre- vs. post-SH: P < 0.001; treatment group: P = 0.010; interaction: P = 0.027). The effect of BDNF treatment on the extent of recovery at SH 7D was not statistically significant, with an average DIAm RMS EMG amplitude of 14 ± 5% of the pre-SH sigh value in BDNF-treated animals compared with 2 ± 1% in aCSF-treated animals (P > 0.05). By SH 14D, treatment with BDNF significantly increased the extent of recovery of inspiratory-related DIAm EMG activity compared with aCSF treated animals; average DIAm RMS EMG amplitude was 22 ± 3% of pre-SH sigh values in BDNF-treated animals compared with 4 ± 3% in the aCSF-treated SH animals (P < 0.05). In the three aCSF-treated SH rats displaying eupneic DIAm EMG activity at SH 14D, average DIAm RMS EMG amplitude was 11 ± 3% of pre-SH sigh values. Although the amplitude of DIAm RMS EMG activity increased in BDNF-treated animals relative to aCSF-treated SH rats, the amplitude in both treatment groups remained lower than pre-SH levels.

During hypoxia-hypercapnia, there was also a statistically significant SH-induced decrease in DIAm RMS EMG amplitude that was ameliorated by BDNF treatment (2-way ANOVA; overall: P < 0.001; pre- vs. post-SH: P < 0.005; treatment group: P < 0.01; interaction: P < 0.01). BDNF treatment significantly increased DIAm RMS EMG amplitude at SH 14D compared with aCSF treatment; DIAm RMS EMG amplitude was 51 ± 6% of the pre-SH sigh value in BDNF-treated animals compared with 15 ± 6% of the pre-SH sigh value in aCSF-treated animals (P < 0.05). BDNF treatment increased DIAm RMS EMG amplitude such that it approached pre-SH levels, whereas aCSF-treated animals continued to have lower DIAm RMS EMG amplitudes at SH 14D compared with pre-SH levels (P < 0.05). BDNF treatment increased DIAm RMS EMG amplitude during hypoxia-hypercapnia in 4 of 8 animals compared with 0 of 7 aCSF-treated SH animals (P = 0.042; Fig. 3).

Compared with pre-SH values, there were no differences in DIAm RMS EMG amplitudes during sighs at SH 7D or 14D or with BDNF treatment (2-way ANOVA; overall: P = 0.255; pre- vs. post-SH: P = 0.838; treatment group: P = 0.044; interaction: P = 0.326). At SH 7D, the amplitude of DIAm RMS EMG activity during sighs was 121 ± 46% of the pre-SH sigh in BDNF-treated animals compared with 58 ± 9% of the pre-SH sigh in aCSF-treated SH animals. Similarly, at SH 14D, the amplitude of DIAm RMS EMG activity during sighs was 99 ± 12% of the pre-SH sigh value in BDNF-treated animals compared with 75 ± 12% of the pre-SH sigh value in aCSF-treated SH animals. In addition, the incidence of sighs was 0.2 min−1 before SH and did not change with SH or with BDNF treatment. The extent of recovery of DIAm EMG activity during sighs at SH 14D varied across animals, with 3 of 8 BDNF-treated animals showing an increase in activity compared with 1 of 7 aCSF-treated animals (P = 0.310; Fig. 3).

Compared with pre-SH values, there were no differences in DIAm RMS EMG amplitudes during airway occlusion following SH or with BDNF treatment (Fig. 2; 2-way ANOVA; overall: P = 0.217; pre- vs. post-SH: P = 0.335; treatment group: P = 0.234; interaction: P = 0.124). The amplitude of DIAm RMS EMG activity during airway occlusion was 132 ± 14% of the pre-SH sigh value in BDNF-treated animals compared with 91 ± 13% of the pre-SH sigh value in the aCSF-treated SH animals. The extent of recovery of DIAm EMG after SH during airway occlusion varied across animals, with 4 of 6 BDNF-treated animals displaying an increase compared with 2 of 7 aCSF-treated animals (P = 0.170; Fig. 3).

Compared with pre-SH values, there were no differences in DIAm RMS EMG amplitude during sneezing following SH or with BDNF treatment (Fig. 2; 2-way ANOVA; overall: P = 0.070; pre-vs. post-SH: P = 0.949; treatment group: P = 0.033; interaction: P = 0.103). The amplitude of DIAm RMS EMG activity during sneezing was 164 ± 15% of the pre-SH sigh in BDNF-treated animals compared with 99 ± 17% of the pre-SH sigh value in the aCSF-treated SH animals. The extent of recovery of DIAm EMG after SH during sneezing varied across animals with 5 of 8 BDNF-treated animals displaying an increase compared with 1 of 7 aCSF-treated animals (P = 0.057; Fig. 3).

Changes in contralateral DIAm EMG activity following SH.

During eupnea, sigh, airway occlusion, and sneezing, there were no differences in contralateral DIAm RMS EMG amplitude across groups (Fig. 2). However, during hypoxia-hypercapnia, there was a statistically significant increase in DIAm RMS EMG amplitude following SH (Fig. 2; 2-way ANOVA; overall: P = 0.019; pre- vs. post-SH: P = 0.018; treatment group: P = 0.166; interaction: P = 0.105). The amplitude of contralateral DIAm RMS EMG activity during hypoxia-hypercapnia at SH 14D was 101 ± 12% of the pre-SH sigh value compared with 48 ± 5% of the sigh value before SH. Changes in the amplitude of the contralateral DIAm RMS EMG activity across motor behaviors and treatment conditions varied widely across animals (Fig. 3).

Ventilatory parameters.

Respiratory rate, burst duration, and duty cycle were measured from contralateral DIAm EMG recordings during eupnea and hypoxia-hypercapnia at pre-SH and SH 14D (Table 1). Respiratory rate was unaffected by SH and/or BDNF treatment during eupnea (repeated-measures ANOVA; pre- vs. post-SH: P = 0.07; treatment group: P = 0.41; interaction: P = 0.12) or hypoxia-hypercapnia (pre- vs. post-SH: P = 0.28; treatment group: P = 0.66; interaction: P = 0.36). Burst duration was also unaffected by SH or BDNF treatment during eupnea (pre- vs. post-SH: P = 0.53; treatment group: P = 0.58; interaction: P = 0.07) or hypoxia-hypercapnia (pre- vs. post-SH: P = 0.42; treatment group: P = 0.16; interaction: P = 0.71). Accordingly, duty cycle was unaffected by SH and/or treatment during eupnea (pre- vs. post-SH: P = 0.24; treatment group: P = 0.71; interaction: P = 0.99) or hypoxia-hypercapnia (pre- vs. post-SH: P = 0.12; treatment group: P = 0.61; interaction: P = 0.51).

Table 1.

Ventilatory parameters across ventilatory behaviors before and after unilateral cervical spinal cord hemisection at C2 (SH)

Experimental Groups
Ventilatory Parameter aCSF pre-SH aCSF SH 14D BDNF pre-SH BDNF SH 14D
Eupnea
Respiratory rate, min−1 81 ± 7 83 ± 9 75 ± 7 101 ± 5
Burst duration, ms 339 ± 44 369 ± 23 369 ± 22 309 ± 14
Duty cycle,% 44 ± 4 50 ± 6 45 ± 4 51 ± 3
Hypoxia-hypercapnia
Respiratory rate, min−1 100 ± 10 101 ± 9 96 ± 5 111 ± 4
Burst duration, ms 316 ± 20 333 ± 21 294 ± 11 300 ± 19
Duty cycle,% 51 ± 3 54 ± 3 47 ± 3 55 ± 3
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Values are ventilatory parameters before and after unilateral spinal cord hemisection at C2 (SH) during eupnea and hypoxia-hypercapnia behaviors. Respiratory rate, burst duration, and duty cycle were measured from contralateral DIAm EMG recordings before (pre-SH) and 14 days after SH (SH 14D). There were no significant differences in ventilatory parameters during either behavior between animals treated with aCSF and BDNF or between pre-SH and SH 14D recordings. Data are means ± SE.

DISCUSSION

The results of the present study indicate that following incomplete unilateral cervical spinal cord injury, intrathecal BDNF treatment enhances recovery of DIAm activity during ventilatory behaviors (eupnea and hypoxia-hypercapnia), with no measurable effect during higher force, nonventilatory motor behaviors. The critical role of BDNF/TrkB signaling at phrenic motor neurons in promoting recovery of ventilatory behaviors is consistent with findings of previous studies from our laboratory (Gransee et al. 2013, 2015; Mantilla et al. 2013a, 2013b; Martinez-Galvez et al. 2016). However, it is important to note that spontaneous recovery of ipsilateral DIAm EMG activity after SH during ventilatory behaviors is incomplete, whereas there is little effect of SH on nonventilatory behaviors at 14 days post-SH. These results suggest that impact of SH and BDNF treatment varies across motor behaviors of the DIAm. It is possible that these different motor behaviors are mediated by descending synaptic inputs that vary in the extent of laterality (i.e., the extent of unilateral vs. bilateral input to phrenic motor neurons) or by local propriospinal input. It is likely that the effect of BDNF treatment is to increase synaptic strength, by increasing either the excitability of phrenic motor neurons or the release of excitatory neurotransmitters. Thus the apparent effect of BDNF treatment at phrenic motor neurons may be limited to rhythmic ventilatory DIAm behaviors mediated by predominantly ipsilateral descending synaptic input that is submaximal. As synaptic drive increases in more forceful, nonventilatory motor behaviors, the opportunity to increase synaptic strength via BDNF/TrkB signaling may be reduced.

Diaphragm motor unit recruitment during different motor behaviors.

Motor units are the final element of motor control of skeletal muscles and include a motor neuron and the group of muscle fibers it innervates (Liddell and Sherrington 1925; Mantilla et al. 2010, 2014b; Sieck 1988; Sieck and Fournier 1989). Recruitment of DIAm motor units determines the range of motor behaviors that can be accomplished; from lower force, rhythmic ventilatory behaviors to higher force, nonventilatory behaviors (Sieck and Fournier 1989). It is likely that DIAm motor unit recruitment occurs in an orderly fashion such that more fatigue-resistant motor units (type S and type FR) are recruited first to accomplish sustained ventilatory behaviors, whereas more fatigable motor units (type FInt and type FF) are recruited only during more forceful, nonventilatory behaviors (Mantilla et al. 2010; Seven et al. 2014; Sieck 1988, 1994; Sieck and Fournier 1989). Thus eupnea and breathing in response to hypoxia-hypercapnia are likely to require the recruitment of only fatigue resistant type S and FR motor units in the rat DIAm. More forceful breathing efforts against an occluded airway require additional recruitment of nearly all type FInt motor units, and only during sneezing is the recruitment of type FF units required. This model of DIAm motor unit activation assumes orderly recruitment based primarily on motor neuron size and the corresponding intrinsic electrophysiological properties of motor neurons (Henneman 1957; Henneman et al. 1965). Accordingly, with similar excitatory synaptic input, smaller phrenic motor neurons are recruited first because of their lower membrane capacitance and higher membrane resistance, followed by larger motor neurons with higher membrane capacitance and lower membrane resistance.

Spontaneous recovery of DIAm EMG activity after SH.

Unilateral spinal hemisection at C2 is a well-established model of spinal cord injury, which abolishes descending bulbospinal excitatory input to ipsilateral phrenic motor neurons (Fuller et al. 2006; Goshgarian 2003; Gransee et al. 2013, 2015; Mantilla et al. 2013a, 2013b; Martinez-Galvez et al. 2016; Miyata et al. 1995; Porter 1895; Prakash et al. 1995, 1999; Zhan et al. 1997). The predominant descending premotor synaptic drive to phrenic motor neurons mediating inspiratory efforts is ipsilateral with a smaller contralateral input (Ellenberger and Feldman 1988, 1990). After SH, the predominant ipsilateral descending input to phrenic motor neurons for inspiration is removed, phrenic motor neurons are no longer activated, and the DIAm is paralyzed during eupnea on that side. Recovery of rhythmic ipsilateral DIAm activity following SH depends on strengthening the residual contralateral premotor input to ipsilateral phrenic motor neurons through either pre- or postsynaptic adaptations. Although progressive spontaneous recovery of rhythmic eupneic DIAm activity occurs over time following SH, the extent of recovery is minimal and does not return to preinjury levels (up to 42 days after SH) (Mantilla et al. 2013b; Nantwi et al. 1999). In the present study, the effect of SH induced removal of ipsilateral descending drive to phrenic motor neurons was confirmed by the absence of DIAm EMG activity at the time of surgery and at 3 days after SH. Thereafter, spontaneous functional recovery of rhythmic ipsilateral eupneic DIAm EMG activity occurred after SH, with ∼20% of animals displaying some recovery at 7 days and ∼40% at 14 days after SH, in agreement with previous studies (Gransee et al. 2013, 2015; Mantilla et al. 2013a, 2013b; Martinez-Galvez et al. 2016). Increasing inspiratory-related drive by hypoxia-hypercapnia increased DIAm EMG activity after SH, but the extent of recovery was still limited.

In mammals, respiratory activity is generated and modulated in a region of the ventral respiratory group (VRG) called pre-Bötzinger complex (preBötC; Smith et al. 1991), which is active during different motor behaviors including eupnea, hypoxia-hypercapnia, and sighs. Indeed, it was recently reported that sighs are enhanced inspiratory efforts that result from the projection of a small population of ∼200 peptidergic neurons in the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) to the preBötC, which then convert a normal breath to a sigh (Li et al. 2016). Alternatively, sigh generation may involve a reconfiguration of the activity of neurons in the preBötC (Lieske et al. 2000). In both cases, the anatomical source of premotor output to phrenic motor neurons would be similar but would vary in strength (i.e., inspiratory drive). Similarly, the source of enhanced premotor drive during hypoxia-hypercapnia would be the same. Thus SH, by eliminating the predominant ipsilateral premotor drive to phrenic motor neurons, would reduce the amplitude of DIAm RMS EMG activity during both ventilatory (eupnea and hypoxia-hypercapnia) and sigh behaviors.

Premotor input for higher force, nonventilatory behaviors could vary (Mantilla et al. 2014b). Indeed, it is possible that pattern generators and descending bulbospinal pathways mediating each of the higher force, nonventilatory behaviors (sigh, occlusion, and sneezing) may be different. The neural mechanism(s) that underlie the increased premotor drive to phrenic motor neurons during higher force, nonventilatory behaviors, and the extent of bilateral distribution of this input, is largely unknown.

In this study, we used chronic recordings of DIAm EMG activity, which we previously demonstrated are stable over several weeks when referenced to DIAm EMG amplitude during sighs (Mantilla et al. 2011). If the distribution of descending medullary premotor input differs between ventilatory and higher force, nonventilatory behaviors, the effect of removal of ipsilateral input via SH will vary between these motor behaviors. The amplitude of the ipsilateral DIAm EMG activity during eupnea and hypoxia-hypercapnia (normalized to pre-SH sigh) was lower after SH compared with preinjury levels. This is consistent with a reduction in the contribution of the ipsilateral premotor input to overall ventilatory activity after SH. In contrast, the extent of DIAm EMG activity during more forceful, nonventilatory behaviors was not measurably affected by SH. This may reflect differences in the distribution of descending premotor input to phrenic motor neurons for these behaviors, with a greater contralateral input mediating more forceful, nonventilatory behaviors.

Based on the results of the present study, it is clear that recovery of ipsilateral DIAm EMG activity occurs across all motor behaviors following SH, albeit to varying extents. This recovery most likely depends on strengthening the residual contralateral premotor input to ipsilateral phrenic motor neurons. These results are consistent with almost all previous studies that have examined only spontaneous recovery of rhythmic ventilatory behaviors of the DIAm.

Impact of BDNF treatment on recovery of DIAm EMG activity.

In previous studies, we found that recovery of ipsilateral eupneic DIAm EMG activity after SH is enhanced either by intrathecally delivering BDNF to the area of the spinal cord containing phrenic motor neurons, as in the present study (Mantilla et al. 2013a), or via intraspinal transplantation of mesenchymal stem cells engineered to release BDNF (Gransee et al. 2015). In both cases, BDNF delivery is not restricted to phrenic motor neurons but may affect other cells in the spinal cord segments containing the phrenic motor pool (e.g., neighboring glia). However, our laboratory has provided substantial evidence demonstrating an effect of BDNF/TrkB signaling at phrenic motoneurons in recovery after SH (Gransee et al. 2013, 2016; Mantilla et al. 2013a; Martinez-Galvez et al. 2016). It is possible that the pre- and/or postsynaptic effects of BDNF/TrkB signaling vary across phrenic motor neurons, thereby differentially affecting premotor synaptic drive or motor neuron excitability. The results of this study suggest that for ventilatory behaviors, premotor drive for breathing is predominantly ipsilateral, with limited spontaneous recovery that can be enhanced via BDNF/TrkB signaling, presumably at smaller phrenic motor neurons. In contrast, higher force, nonventilatory behaviors display minimal effects of SH, perhaps because premotor drive to phrenic motor neurons is more bilaterally distributed for these behaviors. In this case, postsynaptically enhancing BDNF/TrkB signaling may not exert as pronounced an effect compared with ventilatory behaviors; i.e., DIAm RMS EMG amplitude is already saturated and further effects cannot be seen.

Conclusions.

This study demonstrates that after SH, there are differences in the spontaneous recovery of DIAm EMG activity during ventilatory compared with more forceful, nonventilatory motor behaviors. Furthermore, our results support our previous finding that BDNF/TrkB signaling at phrenic motor neurons plays a critical role in the functional recovery of rhythmic DIAm activity after SH (Gransee et al. 2013, 2015; Mantilla et al. 2013a, 2014a; Martinez-Galvez et al. 2016). However, the effect of intrathecal BDNF treatment appears to depend on the motor behavior, reflecting differences in ipsilateral activity in this model of incomplete unilateral spinal cord injury. Accordingly, BDNF treatment at the level of the phrenic motor neuron pool enhances recovery of ipsilateral DIAm activity following unilateral SH, exerting main effects on recovery of ventilatory but not higher force, nonventilatory behaviors.

GRANTS

This study was supported by National Heart, Lung, and Blood Institute Grant R01 HL96750 and Mayo Clinic.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

V.H.-T., H.M.G., Y.W., and W.-Z.Z. performed experiments; V.H.-T., H.M.G., C.B.M., Y.W., W.-Z.Z., and G.C.S. analyzed data; V.H.-T., H.M.G., C.B.M., Y.W., and G.C.S. interpreted results of experiments; V.H.-T., H.M.G., and C.B.M. prepared figures; V.H.-T., H.M.G., C.B.M., and G.C.S. drafted manuscript; V.H.-T., H.M.G., C.B.M., and G.C.S. edited and revised manuscript; V.H.-T., H.M.G., C.B.M., Y.W., W.-Z.Z., and G.C.S. approved final version of manuscript.

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