Wen Zhi Zhan

Diaphragm Motor Unit Recruitment in Rats

We hypothesized that considerable force reserve exists for the diaphragm muscle (DIAm) to generate transdiaphragmatic pressures (Pdi) necessary to sustain ventilation. In rats, we measured Pdi and DIAm EMG activity during different ventilatory (eupnea and hypoxia (10% O(2))-hypercapnia (5% CO(2))) and non-ventilatory (airway occlusion and sneezing induced by intranasal capsaicin) behaviors. Compared to maximum Pdi (Pdi(max) generated by bilateral phrenic nerve stimulation), the Pdi generated during eupnea (21+/-2%) and hypoxia-hypercapnia (28+/-4%) were significantly less (p<0.0001) than that generated during airway occlusion (63+/-4%) and sneezing (94+/-5%). The Pdi generated during spontaneous sighs was 62+/-5% of Pdi(max). Relative DIAm EMG activity (root mean square [RMS] amplitude) paralleled the changes in Pdi during different ventilatory and non-ventilatory behaviors (r(2)=0.78; p<0.0001). These results support our hypothesis of a considerable force reserve for the DIAm to accomplish ventilatory behaviors. A model for DIAm motor unit recruitment predicted that ventilatory behaviors would require activation of only fatigue resistant units.

Inactivity-induced remodeling of neuromuscular junctions in rat diaphragmatic muscle.

We hypothesized that inactivity‐induced remodeling of neuromuscular junctions (NMJs) depends on fiber type and the match between muscle fiber and motoneuron (MN) activities. Two inactivity models were studied in rat diaphragmatic muscle: spinal hemisection at C2 (SH), where both diaphragmatic muscle fibers and phrenic MNs were inactive, and tetrodotoxin (TTX) nerve blockade, where only muscle fibers were inactive. After 2 weeks of inactivity, there was increased number of pre‐ and postsynaptic branches (fragmentation) of NMJs at type IIx/b fibers in both models. In addition, planar NMJ areas at type IIx/b fibers in the SH model were enlarged. In contrast, NMJs at type I and IIa fibers were unaffected in both SH and TTX models. Functionally, neuromuscular transmission in diaphragmatic muscle fibers improved in the SH model, but worsened in the TTX model, compared to controls. These results suggest that NMJ remodeling depends on the level of MN activity. The relative preservation of NMJs at type I and IIa fibers suggests a potential for recovery from diaphragmatic paralysis in the clinical setting, at least for respiratory behaviors.

Motoneuron BDNF/TrkB signaling enhances functional recovery after cervical spinal cord injury

A C2 cervical spinal cord hemisection (SH) interrupts descending inspiratory-related drive to phrenic motoneurons located between C3 and C5 in rats, paralyzing the ipsilateral hemidiaphragm muscle. There is gradual recovery of rhythmic diaphragm muscle activity ipsilateral to cervical spinal cord injury over time, consistent with neuroplasticity and strengthening of spared, contralateral descending premotor input to phrenic motoneurons. Brain-derived neurotrophic factor (BDNF) signaling through the tropomyosin related kinase receptor subtype B (TrkB) plays an important role in neuroplasticity following spinal cord injury. We hypothesized that 1) increasing BDNF/TrkB signaling at the level of the phrenic motoneuron pool by intrathecal BDNF delivery enhances functional recovery of rhythmic diaphragm activity after SH, and 2) inhibiting BDNF/TrkB signaling by quenching endogenous neurotrophins with the soluble fusion protein TrkB-Fc or by knocking down TrkB receptor expression in phrenic motoneurons using intrapleurally-delivered siRNA impairs functional recovery after SH. Diaphragm EMG electrodes were implanted bilaterally to verify complete hemisection at the time of SH and 3days post-SH. After SH surgery in adult rats, an intrathecal catheter was placed at C4 to chronically infuse BDNF or TrkB-Fc using an implanted mini-osmotic pump. At 14days post-SH, all intrathecal BDNF treated rats (n=9) displayed recovery of ipsilateral hemidiaphragm EMG activity, compared to 3 out of 8 untreated SH rats (p<0.01). During eupnea, BDNF treated rats exhibited 76±17% of pre-SH root mean squared EMG vs. only 5±3% in untreated SH rats (p<0.01). In contrast, quenching endogenous BDNF with intrathecal TrkB-Fc treatment completely prevented functional recovery up to 14days post-SH (n=7). Immunoreactivity of the transcription factor cAMP response element-binding protein (CREB), a downstream effector of TrkB signaling, increased in phrenic motoneurons following BDNF treatment (n=6) compared to artificial cerebrospinal fluid treatment (n=6; p<0.001). Intrapleural injections of non-sense or TrkB siRNA were administered after SH to specifically target phrenic motoneurons. At 14days post-SH, none out of 9 TrkB siRNA treated rats displayed functional recovery compared to 5 out of 9 non-sense siRNA treated rats. These results indicate that BDNF/TrkB signaling in phrenic motoneuron pool plays a critical role in functional recovery after cervical spinal cord injury.

The effect of denervation on protein synthesis and degradation in adult rat diaphragm muscle

Previous studies showed that unilateral denervation (DNV) of the rat diaphragm muscle (DIAm) results in loss of myosin heavy chain protein by 1 day after DNV. We hypothesize that DNV decreases net protein balance as a result of activation of the ubiquitin-proteasome pathway. In DIAm strips, protein synthesis was measured by incorporation of 3H-Tyr, and protein degradation was measured by Tyr release at 1, 3, 5, 7, and 14 days after DNV. Total protein ubiquitination, caspase-3 expression/activity, and actin fragmentation were analyzed by Western analysis. We found that, at 3 days after DNV, protein synthesis increased by 77% relative to sham controls. Protein synthesis remained elevated at 5 (85%), 7 (53%), and 14 days (123%) after DNV. At 5 days after DNV, protein degradation increased by 43% relative to sham controls and remained elevated at 7 (49%) and 14 days (74%) after DNV. Thus, by 5 days after DNV, net protein balance decreased by 43% compared with sham controls and was decreased compared with sham at 7 (49%) and 14 days (72%) after DNV. Protein ubiquitination increased at 5 days after DNV and remained elevated. DNV had no effect on caspase-3 activity or actin fragmentation, suggesting that the ubiquitin-proteasome pathway rather than caspase-3 activation is important in the DIAm response to DNV. Early loss of contractile proteins, such as myosin heavy chain, is likely the result of selective protein degradation rather than generalized protein breakdown. Future studies should evaluate this selective effect of DNV.

Targeted delivery of TrkB receptor to phrenic motoneurons enhances functional recovery of rhythmic phrenic activity after cervical spinal hemisection.

Progressive recovery of rhythmic phrenic activity occurs over time after a spinal cord hemisection involving unilateral transection of anterolateral funiculi at C2 (SH). Brain-derived neurotrophic factor (BDNF) acting through its full-length tropomyosin related kinase receptor subtype B (TrkB.FL) contributes to neuroplasticity after spinal cord injury, but the specific cellular substrates remain unclear. We hypothesized that selectively targeting increased TrkB.FL expression to phrenic motoneurons would be sufficient to enhance recovery of rhythmic phrenic activity after SH. Several adeno-associated virus (AAV) serotypes expressing GFP were screened to determine specificity for phrenic motoneuron transduction via intrapleural injection in adult rats. GFP expression was present in the cervical spinal cord 3 weeks after treatment with AAV serotypes 7, 8, and 9, but not with AAV2, 6, or rhesus-10. Overall, AAV7 produced the most consistent GFP expression in phrenic motoneurons. SH was performed 3 weeks after intrapleural injection of AAV7 expressing human TrkB.FL-FLAG or saline. Delivery of TrkB.FL-FLAG to phrenic motoneurons was confirmed by FLAG protein expression in the phrenic motor nucleus and human TrkB.FL mRNA expression in microdissected phrenic motoneurons. In all SH rats, absence of ipsilateral diaphragm EMG activity was confirmed at 3 days post-SH, verifying complete interruption of ipsilateral descending drive to phrenic motoneurons. At 14 days post-SH, all AAV7-TrkB.FL treated rats (n = 11) displayed recovery of ipsilateral diaphragm EMG activity compared to 3 out of 8 untreated SH rats (p<0.01). During eupnea, AAV7-TrkB.FL treated rats exhibited 73±7% of pre-SH root mean squared EMG vs. only 31±11% in untreated SH rats displaying recovery (p<0.01). This study provides direct evidence that increased TrkB.FL expression in phrenic motoneurons is sufficient to enhance recovery of ipsilateral rhythmic phrenic activity after SH, indicating that selectively targeting gene expression in spared motoneurons below the level of spinal cord injury may promote functional recovery.

Prolonged C2 spinal hemisection-induced inactivity reduces diaphragm muscle specific force with modest, selective atrophy of type IIx and/or IIb fibers

The diaphragm muscle (DIAm) is critically responsible for sustaining ventilation. Previously we showed in a commonly used model of spinal cord injury, unilateral spinal cord hemisection at C(2) (SH), that there are minimal changes to muscle fiber cross-sectional area (CSA) and fiber type distribution following 14 days of SH-induced ipsilateral DIAm inactivity. In the present study, effects of long-term SH-induced inactivity on DIAm fiber size and force were examined. We hypothesized that prolonged inactivity would not result in substantial DIAm atrophy or force loss. Adult rats were randomized to control or SH groups (n = 34 total). Chronic bilateral DIAm electromyographic (EMG) activity was monitored during resting breathing. Minimal levels of spontaneous recovery of ipsilateral DIAm EMG activity were evident in 42% of SH rats (<25% of preinjury root mean square amplitude). Following 42 days of SH, DIAm specific force was reduced 39%. There was no difference in CSA for type I or IIa DIAm fibers in SH rats compared with age, weight-matched controls (classification based on myosin heavy chain isoform expression). Type IIx and/or IIb DIAm fibers displayed a modest 20% reduction in CSA (P < 0.05). Overall, there were no differences in the distribution of fiber types or the contribution of each fiber type to the total DIAm CSA. These data indicate that reduced specific force following prolonged inactivity of the DIAm is associated with modest, fiber type selective adaptations in muscle fiber size and fiber type distribution.

Chronic assessment of diaphragm muscle EMG activity across motor behaviors

The diaphragm muscle is the main inspiratory muscle in mammals. Quantitative analyses documenting the reliability of chronic diaphragm EMG recordings are lacking. Assessment of ventilatory and non-ventilatory motor behaviors may facilitate evaluating diaphragm EMG activity over time. We hypothesized that normalization of diaphragm EMG amplitude across behaviors provides stable and reliable parameters for longitudinal assessments of diaphragm activity. We found that diaphragm EMG activity shows substantial intra-animal variability over 6 weeks, with coefficient of variation (CV) for different behaviors ∼ 29-42%. Normalization of diaphragm EMG activity to near maximal behaviors (e.g., deep breathing) reduced intra-animal variability over time (CV ∼ 22-29%). Plethysmographic measurements of eupneic ventilation were also stable over 6 weeks (CV ∼ 13% for minute ventilation). Thus, stable and reliable measurements of diaphragm EMG activity can be obtained longitudinally using chronically implanted electrodes by examining multiple motor behaviors. By quantitatively determining the reliability of longitudinal diaphragm EMG analyses, we provide an important tool for evaluating the progression of diseases or injuries that impair ventilation.

Structure-activity relationships in rodent diaphragm muscle fibers vs. neuromuscular junctions

The diaphragm muscle (DIAm) is a highly active muscle of mixed fiber type composition. We hypothesized that consistent with greater activation history and proportion of fatigue-resistant fibers, neuromuscular transmission failure is lower in the mouse compared to the rat DIAm, and that neuromuscular junction (NMJ) morphology will match their different functional demands. Minute ventilation and duty cycle were higher in the mouse than in the rat. The proportion of fatigue-resistant fibers was similar in the rat and mouse; however the contribution of fatigue-resistant fibers to total DIAm mass was higher in the mouse. Neuromuscular transmission failure was less in mice than in rats. Motor end-plate area differed across fibers in rat but not in mouse DIAm, where NMJs displayed greater complexity overall. Thus, differences across species in activation history and susceptibility to neuromuscular transmission failure are reflected in the relative contribution of fatigue resistant muscle fibers to total DIAm mass, but not in type-dependent morphological differences at the NMJ.

Localized Delivery of Brain-Derived Neurotrophic Factor-Expressing Mesenchymal Stem Cells Enhances Functional Recovery following Cervical Spinal Cord Injury

Neurotrophins, such as brain-derived neurotrophic factor (BDNF), are important in modulating neuroplasticity and promoting recovery after spinal cord injury. Intrathecal delivery of BDNF enhances functional recovery following unilateral spinal cord hemisection (SH) at C2, a well-established model of incomplete cervical spinal cord injury. We hypothesized that localized delivery of BDNF-expressing mesenchymal stem cells (BDNF-MSCs) would promote functional recovery of rhythmic diaphragm activity after SH. In adult rats, bilateral diaphragm electromyographic (EMG) activity was chronically monitored to determine evidence of complete SH at 3 days post-injury, and recovery of rhythmic ipsilateral diaphragm EMG activity over time post-SH. Wild-type, bone marrow-derived MSCs (WT-MSCs) or BDNF-MSCs (2×10(5) cells) were injected intraspinally at C2 at the time of injury. At 14 days post-SH, green fluorescent protein (GFP) immunoreactivity confirmed MSCs presence in the cervical spinal cord. Functional recovery in SH animals injected with WT-MSCs was not different from untreated SH controls (n=10; overall, 20% at 7 days and 30% at 14 days). In contrast, functional recovery was observed in 29% and 100% of SH animals injected with BDNF-MSCs at 7 days and 14 days post-SH, respectively (n=7). In BDNF-MSCs treated SH animals at 14 days, root-mean-squared EMG amplitude was 63±16% of the pre-SH value compared with 12±9% in the control/WT-MSCs group. We conclude that localized delivery of BDNF-expressing MSCs enhances functional recovery of diaphragm muscle activity following cervical spinal cord injury. MSCs can be used to facilitate localized delivery of trophic factors such as BDNF in order to promote neuroplasticity following spinal cord injury.

Phrenic motoneuron expression of serotonergic and glutamatergic receptors following upper cervical spinal cord injury

Following cervical spinal cord injury at C(2) (SH hemisection model) there is progressive recovery of phrenic activity. Neuroplasticity in the postsynaptic expression of neurotransmitter receptors may contribute to functional recovery. Phrenic motoneurons express multiple serotonergic (5-HTR) and glutamatergic (GluR) receptors, but the timing and possible role of these different neurotransmitter receptor subtypes in the neuroplasticity following SH are not clear. The current study was designed to test the hypothesis that there is an increased expression of serotonergic and glutamatergic neurotransmitter receptors within phrenic motoneurons after SH. In adult male rats, phrenic motoneurons were labeled retrogradely by intrapleural injection of Alexa 488-conjugated cholera toxin B. In thin (10μm) frozen sections of the spinal cord, fluorescently-labeled phrenic motoneurons were visualized for laser capture microdissection (LCM). Using quantitative real-time RT-PCR in LCM samples, the time course of changes in 5-HTR and GluR mRNA expression was determined in phrenic motoneurons up to 21 days post-SH. Expression of 5-HTR subtypes 1b, 2a and 2c and GluR subtypes AMPA, NMDA, mGluR1 and mGluR5 was evident in phrenic motoneurons from control and SH rats. Phrenic motoneuron expression of 5-HTR2a increased ~8-fold (relative to control) at 14 days post-SH, whereas NMDA expression increased ~16-fold by 21-days post-SH. There were no other significant changes in receptor expression at any time post-SH. This is the first study to systematically document changes in motoneuron expression of multiple neurotransmitter receptors involved in regulation of motoneuron excitability. By providing information on the neuroplasticity of receptors expressed in a motoneuron pool that is inactivated by a higher-level spinal cord injury, appropriate pharmacological targets can be identified to alter motoneuron excitability.