Enhanced Diaphragm Muscle Function upon Satellite Cell Transplantation in Dystrophic Mice.

The diaphragm muscle is essential for breathing, and its dysfunctions can be fatal. Many disorders affect the diaphragm, including muscular dystrophies. Despite the clinical relevance of targeting the diaphragm, there have been few studies evaluating diaphragm function following a given experimental treatment, with most of these involving anti-inflammatory drugs or gene therapy. Cell-based therapeutic approaches have shown success promoting muscle regeneration in several mouse models of muscular dystrophy, but these have focused mainly on limb muscles. Here we show that transplantation of as few as 5000 satellite cells directly into the diaphragm results in consistent and robust myofiber engraftment in dystrophin- and fukutin-related protein-mutant dystrophic mice. Transplanted cells also seed the stem cell reservoir, as shown by the presence of donor-derived satellite cells. Force measurements showed enhanced diaphragm strength in engrafted muscles. These findings demonstrate the feasibility of cell transplantation to target the diseased diaphragm and improve its contractility.

Chloroquine impairs maximal transdiaphragmatic pressure generation in old mice.

Aging results in increased neuromuscular transmission failure and denervation of the diaphragm muscle, as well as decreased force generation across a range of motor behaviors. Increased risk for respiratory complications in old age is a major health problem. Aging impairs autophagy, a tightly regulated multistep process responsible for clearing misfolded or aggregated proteins and damaged organelles. In motor neurons, aging-related autophagy impairment may contribute to deficits in neurotransmission, subsequent muscle atrophy, and loss of muscle force. Chloroquine is commonly used to inhibit autophagy. We hypothesized that chloroquine decreases transdiaphragmatic pressure (Pdi) in mice. Old mice (16-28 mo old; n = 26) were randomly allocated to receive intraperitoneal chloroquine (50 mg/kg) or vehicle 4 h before measuring Pdi during eupnea, hypoxia (10% O2)-hypercapnia (5% CO2) exposure, spontaneous deep breaths ("sighs"), and maximal activation elicited by bilateral phrenic nerve stimulation (Pdimax). Pdi amplitude and ventilatory parameters across experimental groups and behaviors were evaluated using a mixed linear model. There were no differences in Pdi amplitude across treatments during eupnea (∼8 cm H2O), hypoxia-hypercapnia (∼10 cm H2O), or sigh (∼36 cm H2O), consistent with prior studies documenting a lack of aging effects on ventilatory behaviors. In vehicle and chloroquine-treated mice, average Pdimax was 61 and 46 cm H2O, respectively. Chloroquine decreased Pdimax by 24% compared to vehicle (P < 0.05). There were no sex or age effects on Pdi in older mice. The observed decrease in Pdimax suggests aging-related susceptibility to impairments in autophagy, consistent with the effects of chloroquine on this important homeostatic process.NEW & NOTEWORTHY Recent findings suggest that autophagy plays a role in the development of aging-related neuromuscular dysfunction; however, the contribution of autophagy impairment to the maintenance of diaphragm force generation in old age is unknown. This study shows that in old mice, chloroquine administration decreases maximal transdiaphragmatic pressure generation. These chloroquine effects suggest a susceptibility to impairments in autophagy in old age.

Electrophysiological effects of BDNF and TrkB signaling at type-identified diaphragm neuromuscular junctions.

Previous studies show that synaptic quantal release decreases during repetitive stimulation, i.e., synaptic depression. Neurotrophin brain-derived neurotrophic factor (BDNF) enhances neuromuscular transmission via activation of tropomyosin-related kinase receptor B (TrkB). We hypothesized that BDNF mitigates synaptic depression at the neuromuscular junction and that the effect is more pronounced at type IIx and/or IIb fibers compared to type I or IIa fibers given the more rapid reduction in docked synaptic vesicles with repetitive stimulation. Rat phrenic nerve-diaphragm muscle preparations were used to determine the effect of BDNF on synaptic quantal release during repetitive stimulation at 50 Hz. An ∼40% decline in quantal release was observed during each 330-ms duration train of nerve stimulation (intratrain synaptic depression), and this intratrain decline was observed across repetitive trains (20 trains at 1/s repeated every 5 min for 30 min for 6 sets). BDNF treatment significantly enhanced quantal release at all fiber types (P < 0.001). BDNF treatment did not change release probability within a stimulation set but enhanced synaptic vesicle replenishment between sets. In agreement, synaptic vesicle cycling (measured using FM4-64 fluorescence uptake) was increased following BDNF [or neurotrophin-4 (NT-4)] treatment (∼40%; P < 0.05). Conversely, inhibiting BDNF/TrkB signaling with the tyrosine kinase inhibitor K252a and TrkB-IgG (which quenches endogenous BDNF or NT-4) decreased FM4-64 uptake (∼34% across fiber types; P < 0.05). The effects of BDNF were generally similar across all fiber types. We conclude that BDNF/TrkB signaling acutely enhances presynaptic quantal release and thereby may serve to mitigate synaptic depression and maintain neuromuscular transmission during repetitive activation.NEW & NOTEWORTHY Neurotrophin brain-derived neurotrophic factor (BDNF) enhances neuromuscular transmission via activation of tropomyosin-related kinase receptor B (TrkB). Rat phrenic nerve-diaphragm muscle preparations were used to determine the rapid effect of BDNF on synaptic quantal release during repetitive stimulation. BDNF treatment significantly enhanced quantal release at all fiber types. BDNF increased synaptic vesicle cycling (measured using FM4-64 fluorescence uptake); conversely, inhibiting BDNF/TrkB signaling decreased FM4-64 uptake.

CTB-targeted protocells enhance ability of lanthionine ketenamine analogs to induce autophagy in motor neuron-like cells.

Impaired autophagy, a cellular digestion process that eliminates proteins and damaged organelles, has been implicated in neurodegenerative diseases, including motor neuron disorders. Motor neuron targeted upregulation of autophagy may serve as a promising therapeutic approach. Lanthionine ketenamine (LK), an amino acid metabolite found in mammalian brain tissue, activates autophagy in neuronal cell lines. We hypothesized that analogs of LK can be targeted to motor neurons using nanoparticles to improve autophagy flux. Using a mouse motor neuron-like hybrid cell line (NSC-34), we tested the effect of three different LK analogs on autophagy modulation, either alone or loaded in nanoparticles. For fluorescence visualization of autophagy flux, we used a mCherry-GFP-LC3 plasmid reporter. We also evaluated protein expression changes in LC3-II/LC3-I ratio obtained by western blot, as well as presence of autophagic vacuoles per cell obtained by electron microscopy. Delivering LK analogs with targeted nanoparticles significantly enhanced autophagy flux in differentiated motor neuron-like cells compared to LK analogs alone, suggesting the need of a delivery vehicle to enhance their efficacy. In conclusion, LK analogs loaded in nanoparticles targeting motor neurons constitute a promising treatment option to induce autophagy flux, which may serve to mitigate motor neuron degeneration/loss and preserve motor function in motor neuron disease.

Automated evaluation of respiratory signals to provide insight into respiratory drive.

The diaphragm muscle (DIAm) is the primary inspiratory muscle in mammals and is highly active throughout life displaying rhythmic activity. The repetitive activation of the DIAm (and of other muscles driven by central pattern generator activity) presents an opportunity to analyze these physiological data on a per-event basis rather than pooled on a per-subject basis. The present study highlights the development and implementation of a graphical user interface-based algorithm using an analysis of critical points to detect the onsets and offsets of individual respiratory events across a range of motor behaviors, thus facilitating analyses of within-subject variability. The algorithm is designed to be robust regardless of the signal type (e.g., EMG or transdiaphragmatic pressure). Our findings suggest that this approach may be particularly beneficial in reducing animal numbers in certain types of studies, for assessments of perturbation studies where the effects are relatively small but potentially physiologically meaningful, and for analyses of respiratory variability.

Acute intrathecal BDNF enhances functional recovery after cervical spinal cord injury in rats.

Unilateral C2 hemisection (C2SH) disrupts descending inspiratory-related drive to phrenic motor neurons and thus, silences rhythmic diaphragm muscle (DIAm) activity. There is gradual recovery of rhythmic DIAm EMG activity over time post-C2SH, consistent with neuroplasticity, which is enhanced by chronic (2 wk) intrathecal BDNF treatment. In the present study, we hypothesized that acute (30 min) intrathecal BDNF treatment also enhances recovery of DIAm EMG activity after C2SH. Rats were implanted with bilateral DIAm EMG electrodes to verify the absence of ipsilateral eupneic DIAm EMG activity at the time of C2SH and at 3 days post-C2SH. In those animals displaying no recovery of DIAm EMG activity after 28 days (n = 7), BDNF was administered intrathecally (450 mcg) at C4. DIAm EMG activity was measured continuously both before and for 30 min after BDNF treatment, during eupnea, hypoxia-hypercapnia, and spontaneous sighs. Acute BDNF treatment restored eupneic DIAm EMG activity in all treated animals to an amplitude that was 78% ± 9% of pre-C2SH root mean square (RMS) (P < 0.001). In addition, acute BDNF treatment increased DIAm RMS EMG amplitude during hypoxia-hypercapnia (P = 0.023) but had no effect on RMS EMG amplitude during sighs. These results support an acute modulatory role of BDNF signaling on excitatory synaptic transmission at phrenic motor neurons after cervical spinal cord injury.NEW & NOTEWORTHY Brain-derived neurotrophic factor (BDNF) plays an important role in promoting neuroplasticity following unilateral C2 spinal hemisection (C2SH). BDNF was administered intrathecally in rats displaying lack of ipsilateral inspiratory-related diaphragm (DIAm) EMG activity after C2SH. Acute BDNF treatment (30 min) restored eupneic DIAm EMG activity in all treated animals to 78% ± 9% of pre-C2SH level. In addition, acute BDNF treatment increased DIAm EMG amplitude during hypoxia-hypercapnia but had no effect on EMG amplitude during sighs.

TrkB signaling contributes to transdiaphragmatic pressure generation in aged mice.

Ventilatory deficits are common in old age and may result from neuromuscular dysfunction. Signaling via the tropomyosin-related kinase receptor B (TrkB) regulates neuromuscular transmission and, in young mice, is important for the generation of transdiaphragmatic pressure (Pdi). Loss of TrkB signaling worsened neuromuscular transmission failure and reduced maximal Pdi, and these effects are similar to those observed in old age. Administration of TrkB agonists such as 7,8-dihydroxyflavone (7,8-DHF) improves neuromuscular transmission in young and old mice (18 mo; 75% survival). We hypothesized that TrkB signaling contributes to Pdi generation in old mice, particularly during maximal force behaviors. Old male and female TrkBF616A mice, with a mutation that induces 1NMPP1-mediated TrkB kinase inhibition, were randomly assigned to systemic treatment with vehicle, 7,8-DHF, or 1NMPP1 1 h before experiments. Pdi was measured during eupneic breathing (room air), hypoxia-hypercapnia (10% O2/5% CO2), tracheal occlusion, spontaneous deep breaths ("sighs"), and bilateral phrenic nerve stimulation (Pdimax). There were no differences in the Pdi amplitude across treatments during ventilatory behaviors (eupnea, hypoxia-hypercapnia, occlusion, or sigh). As expected, Pdi increased from eupnea and hypoxia-hypercapnia (∼7 cm H2O) to occlusion and sighs (∼25 cm H2O), with no differences across treatments. Pdimax was ∼50 cm H2O in the vehicle and 7,8-DHF groups and ∼40 cm H2O in the 1NMPP1 group (F8,74 = 2; P = 0.02). Our results indicate that TrkB signaling is necessary for generating maximal forces by the diaphragm muscle in old mice and are consistent with aging effects of TrkB signaling on neuromuscular transmission.NEW & NOTEWORTHY TrkB signaling is necessary for generating maximal forces by the diaphragm muscle. In 19- to 21-mo-old TrkBF616A mice susceptible to 1NMPP1-induced inhibition of TrkB kinase activity, maximal Pdi generated by bilateral phrenic nerve stimulation was ∼20% lower after 1NMPP1 compared with vehicle-treated mice. Treatment with the TrkB agonist 7,8-dihydroxyflavone did not affect Pdi generation when compared with age-matched mice. Inhibition of TrkB kinase activity did not affect the forces generated during lower force behaviors in old age.

Age-related impairment of autophagy in cervical motor neurons.

Neuromuscular dysfunction is common in old age. Damaged cytoplasmic structures aggregate with aging, especially in post-mitotic cells like motor neurons. Autophagy is a ubiquitous cell process that aids in the clearance of damaged aggregates. Accordingly, we hypothesized that autophagy is impaired in old age, contributing to neuromuscular dysfunction via an effect in motor neurons. Autophagy flux may be impaired as a result of deficits in the initiation, elongation or degradation phases. Changes in the expression levels of core proteins necessary for each of the autophagy phases were evaluated by Western blotting in the cervical spinal cord (segments C2-C6 corresponding to the phrenic motor pool) of adult male and female mice at 6-, 18-, and 24-months of age (reflecting 100%, 90% and 75% survival, respectively). There was no evidence of an effect of age on the expression of the autophagy markers Beclin-1 (Becn-1; initiation), ATG7 and ATG5/12 complex (elongation) or LC3 (elongation/degradation). Reduced p62 expression (a marker of degradation) was evident in the cervical spinal cord of adult mice at 18-months compared to 24-months. Accordingly, expression of LC3 and p62 in motor neurons was analyzed using immunofluorescence and confocal microscopy in separate animals. LC3 and p62 immunoreactivity was evident in the gray matter with minimal expression in the white matter across all age groups. A mixed linear model with animal as a random effect was used to compare relative LC3 and p62 expression in motor neurons to gray matter across age groups. Expression of both LC3 and p62 was higher in choline acetyl transferase (ChAT)-positive motor neurons (~2-3 fold vs. gray matter). Across age groups, there were differences in the relative expression of LC3 (F2,12 = 7.59, p < 0.01) and p62 (F2,12 = 8.00, p < 0.01) in cervical motor neurons. LC3 expression in motor neurons increased ~20% by 24-months of age in both male and female mice. p62 expression in motor neurons increased ~70% by 18-months compared to 6-months with no further changes by 24-months of age in male mice. p62 expression did not change across age groups in female mice, and was ~20% higher than in males. Our findings highlight important changes in autophagy pathways that likely contribute to the development of aging-related neuromuscular dysfunction in mice. At 18-months of age, increased autophagosome clearance (reduced p62 expression) appears to be a global effect not restricted to motor neurons. By 24-months of age, increased expression of LC3 and p62 indicates impaired autophagy with autophagosome accumulation in cervical motor neurons.

Inhibition of TrkB kinase activity impairs transdiaphragmatic pressure generation.

Signaling via the tropomyosin-related kinase receptor subtype B (TrkB) regulates neuromuscular transmission, and inhibition of TrkB kinase activity by 1NMPP1 in TrkBF616A mice worsens neuromuscular transmission failure (NMTF). We hypothesized that acute inhibition of TrkB kinase activity will impair the ability of the diaphragm muscle to produce maximal transdiaphragmatic pressure (Pdi) without impacting the ability to generate forces associated with ventilation, consistent with the greater susceptibility to NMTF in motor units responsible for higher-force nonventilatory behaviors. Adult male and female TrkBF616A mice were injected with 1NMPP1 (n = 8) or vehicle (DMSO; n = 8) 1 h before Pdi measurements during eupneic breathing, hypoxia/hypercapnia (10% O2/5% CO2), tracheal occlusion, spontaneous deep breaths ("sighs") and during maximal activation elicited by bilateral phrenic nerve stimulation. In the vehicle-treated group, Pdi increased from ~10 cmH2O during eupnea and hypoxia/hypercapnia, to ~35 cmH2O during sighs and tracheal occlusion, and to ~65 cm H2O during maximal stimulation. There was no effect of acute 1NMPP1 treatment on Pdi generated during most behaviors, except during maximal stimulation (~30% reduction; P < 0.05). This reduction in maximal Pdi is generally similar to the worsening of NMTF previously reported with TrkB kinase inhibition in rodents. Accordingly, impaired TrkB signaling limits the range of motor behaviors accomplished by the diaphragm muscle and may contribute to neuromuscular dysfunction, primarily by impacting fatigable, higher force-generating motor units.NEW & NOTEWORTHY TrkB signaling plays an important role in maintaining neuromuscular function in the diaphragm muscle and may be necessary to accomplish the various motor behaviors ranging from ventilation to expulsive, behaviors requiring near-maximal forces. This study shows that inhibition of TrkB kinase activity impairs maximal pressure generation by the diaphragm muscle, but the ability to generate the lower pressures required for ventilatory behaviors is not impacted.

Diaphragm muscle sarcopenia into very old age in mice.

Sarcopenia is the age-related decline of skeletal muscle mass and function. Diaphragm muscle (DIAm) sarcopenia may contribute to respiratory complications, a common cause of morbidity and mortality in the elderly. From 6 to 24 months (mo) of age, representing ~100% and ~80% survival in C57BL/6 × 129 male and female mice, there is a significant reduction in DIAm force generation (~30%) and cross-sectional area (CSA) of type IIx and/or IIb muscle fibers (~30%), impacting the ability to perform high force, non-ventilatory behaviors. To date, there is little information available regarding DIAm sarcopenia in very old age groups. The present study examined DIAm sarcopenia in C57BL/6 × 129 male and female mice at 24, 27, and 30 mo, representing ~80%, ~60%, and ~30% survival, respectively. We hypothesized that survival into older ages will show no further worsening of DIAm sarcopenia and functional impairment in 30 mo mice compared to 24 or 27 mo C57BL/6 × 129 mice. Measurements included resting ventilation, transdiaphragmatic pressure (Pdi) generation across a range of motor behaviors, muscle fiber CSA, and proportion of type-identified DIAm fibers. Maximum Pdi and resting ventilation did not change into very old age (from 24 to 30 mo). Type IIx and/or IIb fiber CSA and proportions did not change into very old age. The results of the study support a critical threshold for the reduction in DIAm force and Pdi such that survival into very old age is not associated with evidence of progression of DIAm sarcopenia or impairment in ventilation.

Frequency-dependent lipid raft uptake at rat diaphragm muscle axon terminals.

INTRODUCTION: In motor neurons, cholera toxin B (CTB) binds to the cell-surface ganglioside GM1 and is internalized and transported via structurally unique components of plasma membranes (lipid rafts). METHODS: Lipid raft uptake by axon terminals adjoining type-identified rat diaphragm muscle fibers was investigated using CTB and confocal imaging. RESULTS: Lipid raft uptake increased significantly at higher frequency stimulation (80 Hz), compared with lower frequency (20 Hz) and unstimulated (0 Hz) conditions. The fraction of axon terminal occupied by CTB was ∼45% at 0- or 20-Hz stimulation, and increased to ∼65% at 80 Hz. Total CTB fluorescence intensity also increased (∼20%) after 80-Hz stimulation compared with 0 Hz. DISCUSSION: Evidence of increased lipid raft uptake at high stimulation frequencies supports an important role for lipid raft signaling at rat diaphragm muscle axon terminals, primarily for motor units physiologically activated at the higher frequencies. Muscle Nerve 59:611-611, 2019.

Phrenic motoneuron structural plasticity across models of diaphragm muscle paralysis.

Structural plasticity in motoneurons may be influenced by activation history and motoneuron-muscle fiber interactions. The goal of this study was to examine the morphological adaptations of phrenic motoneurons following imposed motoneuron inactivity while controlling for diaphragm muscle inactivity. Well-characterized rat models were used including unilateral C2 spinal hemisection (SH; ipsilateral phrenic motoneurons and diaphragm muscle are inactive) and tetrodotoxin phrenic nerve blockade (TTX; ipsilateral diaphragm muscle is paralyzed while phrenic motoneuron activity is preserved). We hypothesized that inactivity of phrenic motoneurons would result in a decrease in motoneuron size, consistent with a homeostatic increase in excitability. Phrenic motoneurons were retrogradely labeled by ipsilateral diaphragm muscle injection of fluorescent dextrans or cholera toxin subunit B. Following 2 weeks of diaphragm muscle paralysis, morphological parameters of labeled ipsilateral phrenic motoneurons were assessed quantitatively using fluorescence confocal microscopy. Compared to controls, phrenic motoneuron somal volumes and surface areas decreased with SH, but increased with TTX. Total phrenic motoneuron surface area was unchanged by SH, but increased with TTX. Dendritic surface area was estimated from primary dendrite diameter using a power equation obtained from three-dimensional reconstructed phrenic motoneurons. Estimated dendritic surface area was not significantly different between control and SH, but increased with TTX. Similarly, TTX significantly increased total phrenic motoneuron surface area. These results suggest that ipsilateral phrenic motoneuron morphological adaptations are consistent with a normalization of motoneuron excitability following prolonged alterations in motoneuron activity. Phrenic motoneuron structural plasticity is likely more dependent on motoneuron activity (or descending input) than muscle fiber activity.

Differences in lumbar motor neuron pruning in an animal model of early onset spasticity.

Motor neuron (MN) development in early onset spasticity is poorly understood. For example, spastic cerebral palsy (sCP), the most common motor disability of childhood, is poorly predicted by brain imaging, yet research remains focused on the brain. By contrast, MNs, via the motor unit and neurotransmitter signaling, are the target of most therapeutic spasticity treatments and are the final common output of motor control. MN development in sCP is a critical knowledge gap, because the late embryonic and postnatal periods are not only when the supposed brain injury occurs but also are critical times for spinal cord neuromotor development. Using an animal model of early onset spasticity [ spa mouse (B6.Cg- Glrbspa/J) with a glycine (Gly) receptor mutation], we hypothesized that removal of effective glycinergic neurotransmitter inputs to MNs during development will influence MN pruning (including primary dendrites) and MN size. Spa (Glrb-/-) and wild-type (Glrb+/+) mice, ages 4-9 wk, underwent unilateral retrograde labeling of the tibialis anterior muscle MNs via peroneal nerve dip in tetramethylrhodamine. After 3 days, mice were euthanized and perfused with 4% paraformaldehyde, and the spinal cord was excised and processed for confocal imaging. Spa mice had ~61% fewer lumbar tibialis anterior MNs ( P < 0.01), disproportionately affecting larger MNs. Additionally, a ~23% reduction in tibialis anterior MN somal surface area ( P < 0.01) and a 12% increase in primary dendrites ( P = 0.046) were observed. Thus MN pruning and MN somal surface area are abnormal in early onset spasticity. Fewer and smaller MNs may contribute to the spastic phenotype. NEW & NOTEWORTHY Motor neuron (MN) development in early onset spasticity is poorly understood. In an animal model of early onset spasticity, spa mice, we found ~61% fewer lumbar tibialis anterior MNs compared with controls. This MN loss disproportionately affected larger MNs. Thus number and heterogeneity of the MN pool are decreased in spa mice, likely contributing to the spastic phenotype.

Diaphragm muscle activity across respiratory motor behaviors in awake and lightly anesthetized rats.

Respiratory muscles such as the diaphragm are active across a range of behaviors including ventilation and higher-force behaviors necessary for maintenance of airway patency, and minimal information is available regarding anesthetic effects on the capacity of respiratory muscles to generate higher forces. The purpose of the present study was to determine whether diaphragm EMG activity during lower-force behaviors, such as eupnea and hypoxia-hypercapnia, is differentially affected compared with higher-force behaviors, such as a sigh, in lightly anesthetized animals. In adult male rats, chronically implanted diaphragm EMG electrodes were used to measure the effects of low-dose ketamine (30 mg/kg) and xylazine (3 mg/kg) on root mean square (RMS) EMG amplitude across a range of motor behaviors. A mixed linear model was used to evaluate the effects of ketamine-xylazine anesthesia on peak RMS EMG and ventilatory parameters, with condition (awake vs. anesthetized), behavior (eupnea, hypoxia-hypercapnia, sigh), side (left or right hemidiaphragm), and their interactions as fixed effects and animal as a random effect. Compared with the awake recordings, there was an overall reduction of peak diaphragm RMS EMG across behaviors during anesthesia, but this reduction was more pronounced during spontaneous sighs (which require ~60% of maximal diaphragm force). Respiratory rates and duty cycle during eupnea and hypoxia-hypercapnia were higher in awake compared with anesthetized conditions. These results highlight the importance of identifying anesthetic effects on a range of respiratory motor behaviors, including sighs necessary for maintaining airway patency. NEW & NOTEWORTHY Respiratory muscles accomplish a range of motor behaviors, with forces generated for ventilatory behaviors comprising only a small fraction of their maximal force generating capacity. Induction of anesthesia exerts more robust effects on the higher-force diaphragm motor behaviors such as sighs compared with eupnea. This novel information on effects of low, sedative doses of a commonly used anesthetic combination (ketamine-xylazine) highlights the importance of identifying anesthetic effects on a range of respiratory motor behaviors.

Impact of glutamatergic and serotonergic neurotransmission on diaphragm muscle activity after cervical spinal hemisection.

Incomplete cervical spinal cord hemisection at C2 (SH) disrupts descending excitatory drive to phrenic motoneurons, paralyzing the ipsilateral diaphragm muscle. Spontaneous recovery over time is associated with increased phrenic motoneuron expression of glutamatergic N-methyl-d-aspartate (NMDA) and serotonergic 5-HT2A receptors. We hypothesized that NMDA and 5-HT2A receptor-mediated neurotransmission play a role in ipsilateral diaphragm muscle activity post-SH. Adult male Sprague-Dawley rats were implanted with bilateral diaphragm EMG electrodes for chronic EMG recordings up to 28 days post-SH (SH 28D). The extent of recovery was calculated by peak root-mean-square (RMS) EMG amplitude. In all animals, absence of ipsilateral activity was verified at 3 days post-SH. Diaphragm EMG activity was also recorded during exposure to hypoxia-hypercapnia (10% O2-5% CO2). In SH animals displaying recovery of ipsilateral diaphragm EMG activity at SH 28D, cervical spinal cord segments containing the phrenic motor nucleus (C3-C5) were surgically exposed and either the NMDA receptor antagonist d-2-amino-5-phosphonovalerate (d-AP5; 100 mM, 30 µl) or 5-HT2A receptor antagonist ketanserin (40 mM, 30 µl) was instilled intrathecally. Following d-AP5, diaphragm EMG amplitude was reduced ipsilaterally, during both eupnea (42% of pre-d-AP5 value; P = 0.007) and hypoxia-hypercapnia (31% of pre-d-AP5 value; P = 0.015), with no effect on contralateral EMG activity or in uninjured controls. Treatment with ketanserin did not change ipsilateral or contralateral RMS EMG amplitude in SH animals displaying recovery at SH 28D. Our results suggest that spinal glutamatergic NMDA receptor-mediated neurotransmission plays an important role in ipsilateral diaphragm muscle activity after cervical spinal cord injury.NEW & NOTEWORTHY Spontaneous recovery following C2 spinal hemisection (SH) is associated with increased phrenic motoneuron expression of glutamatergic and serotonergic receptors. In this study, we show that pharmacological inhibition of glutamatergic N-methyl-d-aspartate (NMDA) receptors blunts ipsilateral diaphragm activity post-SH. In contrast, pharmacological inhibition of serotonergic 5-HT2A receptors does not change diaphragm EMG activity post-SH. Our results suggest that NMDA receptor-mediated glutamatergic neurotransmission plays an important role in enhancing rhythmic respiratory-related diaphragm activity after spinal cord injury.

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

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.

Motoneuron glutamatergic receptor expression following recovery from cervical spinal hemisection.

Cervical spinal hemisection at C2 (SH) removes premotor drive to phrenic motoneurons located in segments C3-C5 in rats. Spontaneous recovery of ipsilateral diaphragm muscle activity is associated with increased phrenic motoneuron expression of glutamatergic N-methyl-D-aspartate (NMDA) receptors and decreased expression of α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptors. Glutamatergic receptor expression is regulated by tropomyosin-related kinase receptor subtype B (TrkB) signaling in various neuronal systems, and increased TrkB receptor expression in phrenic motoneurons enhances recovery post-SH. Accordingly, we hypothesize that recovery of ipsilateral diaphragm muscle activity post-SH, whether spontaneous or enhanced by adenoassociated virus (AAV)-mediated upregulation of TrkB receptor expression, is associated with increased expression of glutamatergic NMDA receptors in phrenic motoneurons. Adult male Sprague-Dawley rats underwent diaphragm electromyography electrode implantation and SH surgery. Rats were injected intrapleurally with AAV expressing TrkB or GFP 3 weeks before SH. At 14 days post-SH, the proportion of animals displaying recovery of ipsilateral diaphragm activity increased in AAV-TrkB-treated (9/9) compared with untreated (3/5) or AAV-GFP-treated (4/10; P < 0.027) animals. Phrenic motoneuron NMDA NR1 subunit mRNA expression was approximately fourfold greater in AAV-TrkB- vs. AAV-GFP-treated SH animals (P < 0.004) and in animals displaying recovery vs. those not recovering (P < 0.005). Phrenic motoneuron AMPA glutamate receptor 2 (GluR2) subunit mRNA expression decreased after SH, and, albeit increased in animals displaying recovery vs. those not recovering, levels remained lower than control. We conclude that increased phrenic motoneuron expression of glutamatergic NMDA receptors is associated with spontaneous recovery after SH and enhanced recovery after AAV-TrkB treatment. J. Comp. Neurol. 525:1192-1205, 2017. © 2016 Wiley Periodicals, Inc.

TrkB gene therapy by adeno-associated virus enhances recovery after cervical spinal cord injury.

Unilateral cervical spinal cord hemisection at C2 (C2SH) interrupts descending bulbospinal inputs to phrenic motoneurons, paralyzing the diaphragm muscle. Recovery after C2SH is enhanced by brain derived neurotrophic factor (BDNF) signaling via the tropomyosin-related kinase subtype B (TrkB) receptor in phrenic motoneurons. The role for gene therapy using adeno-associated virus (AAV)-mediated delivery of TrkB to phrenic motoneurons is not known. The present study determined the therapeutic efficacy of intrapleural delivery of AAV7 encoding for full-length TrkB (AAV-TrkB) to phrenic motoneurons 3 days post-C2SH. Diaphragm EMG was recorded chronically in male rats (n=26) up to 21 days post-C2SH. Absent ipsilateral diaphragm EMG activity was verified 3 days post-C2SH. A greater proportion of animals displayed recovery of ipsilateral diaphragm EMG activity during eupnea by 14 and 21 days post-SH after AAV-TrkB (10/15) compared to AAV-GFP treatment (2/11; p=0.031). Diaphragm EMG amplitude increased over time post-C2SH (p<0.001), and by 14 days post-C2SH, AAV-TrkB treated animals displaying recovery achieved 48% of the pre-injury values compared to 27% in AAV-GFP treated animals. Phrenic motoneuron mRNA expression of glutamatergic AMPA and NMDA receptors revealed a significant, positive correlation (r(2)=0.82), with increased motoneuron NMDA expression evident in animals treated with AAV-TrkB and that displayed recovery after C2SH. Overall, gene therapy using intrapleural delivery of AAV-TrkB to phrenic motoneurons is sufficient to promote recovery of diaphragm activity, adding a novel potential intervention that can be administered after upper cervical spinal cord injury to improve impaired respiratory function.

The Impact of Midcervical Contusion Injury on Diaphragm Muscle Function.

Midcervical contusion injuries disrupt descending ipsilateral excitatory bulbospinal projections to phrenic motoneurons, compromising ventilation. We hypothesized that a unilateral contusion injury at C3 versus C5 would differentially impact phrenic activity reflecting more prominent disruption of ipsilateral descending excitatory drive to more caudal segments of the phrenic motor pool with more cranial injuries. Phrenic motoneuron counts and evidence of diaphragm muscle denervation at individual neuromuscular junctions (NMJ) were evaluated at 14 days post-injury after unilateral contusion injury (100 kDynes). Whole body plethysmography and chronic diaphragm EMG were measured before the injury and at 3, 7, and 14 days post-injury. Contusion injuries at either level resulted in a similarly sized cavity. C3 contusion resulted in loss of 39 ± 13% of ipsilateral phrenic motoneurons compared with 13 ± 21% after C5 contusion (p = 0.003). Cervical contusion injuries resulted in diaphragm muscle denervation (C3 contusion: 17 ± 4%; C5 contusion: 7 ± 4%; p = 0.047). The pattern of denervation revealed segmental innervation of the diaphragm muscle, with greater denervation ventrally after C3 contusion and dorsally after C5 contusion. Overall, diaphragm root mean square electromyography activity did not change ipsilaterally after C3 or C5 contusion, but increased contralaterally (∼ 11%) after C3 contusion only on the first day post-injury (p = 0.026). Similarly, there were no significant changes in breathing parameters during eupnea or exposure to hypoxia (10% O2) - hypercapnia (5% CO2) at any time post-injury. Unilateral midcervical contusions minimally impair ventilatory behaviors despite phrenic motoneuron loss and diaphragm muscle denervation.

Functional recovery after cervical spinal cord injury: Role of neurotrophin and glutamatergic signaling in phrenic motoneurons.

Cervical spinal cord injury (SCI) interrupts descending neural drive to phrenic motoneurons causing diaphragm muscle (DIAm) paralysis. Recent studies using a well-established model of SCI, unilateral spinal hemisection of the C2 segment of the cervical spinal cord (SH), provide novel information regarding the molecular and cellular mechanisms of functional recovery after SCI. Over time post-SH, gradual recovery of rhythmic ipsilateral DIAm activity occurs. Recovery of ipsilateral DIAm electromyogram (EMG) activity following SH is enhanced by increasing brain-derived neurotrophic factor (BDNF) in the region of the phrenic motoneuron pool. Delivery of exogenous BDNF either via intrathecal infusion or via mesenchymal stem cells engineered to release BDNF similarly enhance recovery. Conversely, recovery after SH is blunted by quenching endogenous BDNF with the fusion-protein TrkB-Fc in the region of the phrenic motoneuron pool or by selective inhibition of TrkB kinase activity using a chemical-genetic approach in TrkB(F616A) mice. Furthermore, the importance of BDNF signaling via TrkB receptors at phrenic motoneurons is highlighted by the blunting of recovery by siRNA-mediated downregulation of TrkB receptor expression in phrenic motoneurons and by the enhancement of recovery evident following virally-induced increases in TrkB expression specifically in phrenic motoneurons. BDNF/TrkB signaling regulates synaptic plasticity in various neuronal systems, including glutamatergic pathways. Glutamatergic neurotransmission constitutes the main inspiratory-related, excitatory drive to motoneurons, and following SH, spontaneous neuroplasticity is associated with increased expression of ionotropic N-methyl-d-aspartate (NMDA) receptors in phrenic motoneurons. Evidence for the role of BDNF/TrkB and glutamatergic signaling in recovery of DIAm activity following cervical SCI is reviewed.