Citations

Unilateral C2 spinal cord hemisection (C2SH) removes premotor drive to phrenic motoneurons, paralyzing the ipsilateral diaphragm muscle (DIAm). In previous studies, we found that intrathecal brain‐derived neurotrophic factor (BDNF) increases the proportion of rats displaying recovery of ipsilateral DIAm activity over time. We hypothesized that enhancing expression of the tropomyosin‐related kinase receptor subtype B (TrkB) in phrenic motoneurons will promote functional recovery ipsilateral to C2SH. Adult rats were injected intrapleurally with AAV7 expressing TrkB.FL‐FLAG, and 3 weeks later C2SH was performed. A separate group of C2SH animals served as controls. Bilateral DIAm EMG activity was chronically monitored to assess recovery up to 2 weeks after C2SH. In AAV‐treated rats, delivery of TrkB.FL‐FLAG to phrenic motoneurons was evident by the presence of FLAG protein in the phrenic motor nucleus. At 7‐days post‐C2SH, the proportion of rats displaying ipsilateral DIAm EMG activity was 13% and 40% in the untreated control and AAV‐treated groups, respectively. At 14‐days post‐C2SH, all AAV‐treated rats displayed functional recovery compared to 40% of C2SH rats. We conclude that targeted delivery of TrkB.FL to phrenic motoneurons enhances functional recovery of DIAm respiratory activity following upper cervical spinal cord injury. Supported by NIH grant HL96750 and Paralyzed Veterans of America.

Brain‐derived neurotrophic factor (BDNF) acting through full‐length tropomyosin related kinase receptor subtype B (TrkB.FL) plays an important role in neuroplasticity following spinal cord injury. BDNF/TrkB.FL signaling in phrenic motoneurons is both necessary and sufficient to enhance spontaneous recovery of ipsilateral rhythmic phrenic activity after C2 spinal cord hemisection (SH), where premotor drive to phrenic motoneurons is removed. Phrenic motoneuron expression of the excitatory glutamatergic NMDA receptor correlates with the time course of spontaneous functional recovery post‐SH. We hypothesized that increased BDNF/TrkB.FL signaling in phrenic motoneurons enhances recovery of rhythmic phrenic activity after SH via regulation of glutamatergic receptor expression. Rats were injected intrapleurally with AAV7 expressing human TrkB.FL‐FLAG at 3 weeks prior to SH. FLAG was present in the cervical ventral horn region, and AAV7‐GFP expression was specifically in phrenic motoneurons. At 14 days post‐SH, quantitative real‐time RT‐PCR verified human TrkB.FL expression in microdissected phrenic motoneurons. Phrenic motoneuron expression of NMDA increased ~10 fold compared to control, suggesting that increased BDNF/TrkB.FL signaling magnified SH‐induced changes in phrenic motoneuron NMDA expression. Supported by HL96750 and T32‐HL105355, and Paralyzed Veterans of America.

Following unilateral C2 spinal hemisection (SH), descending excitatory drive to phrenic motoneurons is disrupted causing ipsilateral diaphragm muscle paralysis. After SH, there is gradual recovery of phrenic nerve activity reflecting neuroplasticity. We hypothesized that this neuroplasticity is dependent on BDNF signaling via its TrkB receptor and kinase activity. In this study we used TrkB(F616A) knockin mice, that permit rapid and selective inhibition of TrkB kinase activity by 1NMPP1. Adult TrkB(F616A) mice were treated with 1NMPP1 (25 μM) or vehicle in the drinking water starting 3 days post‐SH. At the terminal experiment, bilateral diaphragm EMG was monitored to determine the proportion of animals displaying recovery of ipsilateral phrenic activity. At 3 days post‐SH, no animals displayed ipsilateral diaphragm EMG activity (0/10). At 14 days post‐SH, 40% (6/15) of TrkB(F616A) mice displayed ipsilateral phrenic activity, although EMG amplitude remained substantially below contralateral levels. Following 1NMPP1 treatment, no animals displayed ipsilateral diaphragm EMG activity (0/10; p<0.05). In control, wild‐type mice, 1NMPP1 treatment (for 11 days) had no evident effect on bilateral diaphragm EMG activity (n=7). These results support an essential role for TrkB kinase activity in the spontaneous recovery of rhythmic phrenic activity post spinal cord injury. Supported by NIH grant HL096750.

Cervical spinal hemisection at C2 (SH) paralyzes the diaphragm muscle by interrupting descending bulbospinal pathways to phrenic motoneurons. Signaling via the neurotrophin receptor TrkB in phrenic motoneurons enhances functional recovery after SH. However, it is not clear if the delay in gene expression resulting from adeno‐associated virus (AAV)‐mediated delivery would blunt the therapeutic efficacy of AAV delivered after SH. The goal of the present study was to examine the efficacy of intrapleurally‐delivered AAV7 encoding the full‐length TrkB receptor at 3 days post‐SH. Chronic diaphragm EMG recordings were conducted in adult male Sprague‐Dawley rats confirming absence of ipsilateral activity 3 days after SH. The proportion of animals displaying recovery of ipsilateral diaphragm EMG activity increased over time post‐SH and was greater in those treated with AAV‐TrkB (by 14 days post‐SH: AAV‐TrkB: 10/15 vs. AAV‐GFP: 2/11; p=0.01). Furthermore, peak root‐mean‐square amplitude of the diaphragm EMG activity increased progressively over time post‐SH. During eupnea, animals displaying recovery of ipsilateral diaphragm EMG activity that were treated with AAV‐TrkB achieved 40% of pre‐injury values (vs. 10% in those treated with AAV‐GFP). Targeting phrenic motoneurons via intrapleural delivery of AAV7‐TrkB promotes recovery of diaphragm activity when administered 3 days post‐injury. Grant Funding Source : Supported by R01 HL96750, T32 HL105355 and Mayo Clinic Center for Regenerative Medicine

Upper cervical spinal cord injury (SCI) disrupts descending inspiratory‐related drive to phrenic motoneurons paralyzing the diaphragm muscle. Over time following unilateral C2 hemisection (SH), there is gradual recovery of rhythmic diaphragm activity ipsilaterally, consistent with neuroplasticity. We recently reported that functional recovery after SH is enhanced by chronic intrathecal delivery of BDNF to the region of the phrenic motor neuron pool. BDNF also exerts acute effects on synaptic transmission that may enhance functional recovery. Accordingly, we hypothesized that intrathecal delivery of BDNF to the phrenic motor neuron pool acutely enhances functional recovery post‐SH. Rats were implanted with diaphragm electrodes for chronic EMG recordings post‐SH, verifying absence of ipsilateral eupneic diaphragm EMG activity by 3 and 28 days after SH. In spontaneously breathing, anesthetized animals, BDNF was administered intrathecally (30 μl) at C4 and ipsilateral rhythmic diaphragm EMG activity was restored (~120% of pre‐injury diaphragm root‐mean‐square [RMS] EMG amplitude) within ~30 min of administration. Diaphragm RMS EMG increased further following exposure to hypoxia (10% O 2 )‐hypercapnia (5% CO 2 ) and intranasal capsaicin. These results highlight the acute role of BDNF signaling in the phrenic motor neuron pool in chronically‐injured rats displaying no spontaneous recovery. Grant Funding Source : Supported by NIH grant R01 HL096750.

Motor units of the diaphragm muscle (DIAm) are recruited in an orderly fashion across ventilatory and non‐ventilatory tasks. Spinal hemisection at C2 (C2HS) blocks descending ipsilateral input to phrenic motoneurons and causes DIAm paralysis. With time after C2HS, there is spontaneous recovery of phrenic nerve activity ipsilateral to the injury. We hypothesized that recruitment order of DIAm motor units is maintained following recovery of DIAm electromyographic (EMG) activity post‐C2HS. DIAm EMG activity in adult male rats was recorded chronically using a pair of electrode wires implanted into the midcostal region bilaterally. DIAm EMG activity was strongly correlated to transdiaphragmatic pressure (Pdi) measured using esophageal and gastric balloons across different motor behaviors (Eupnea, hypoxia‐hypercapnia (10%O 2 and 5%CO 2 ), tracheal occlusion, fictive sneezing and bilateral supramaximal phrenic nerve stimulation). Fictive sneezing produced forces comparable to those elicited by phrenic nerve stimulation, and thus DIAm EMG activity during fictive sneezing was considered maximal. Absence of DIAm EMG activity was verified at the time of C2HS and 3 days (3D) post‐C2HS. Over time, a subset of animals displayed functional recovery of rhythmic DIAm EMG activity ipsilateral to C2HS, although DIAm EMG activity was reduced when compared to pre‐injury levels. EMG activity increased across ventilatory and non‐ventilatory tasks associated with increasing drive in C2HS animals displaying functional recovery, supporting a generalized strengthening of contralateral presynaptic drive over time following spinal cord injury.

Upper cervical spinal cord injury disrupts descending inspiratory‐related drive to phrenic motoneurons (PMN) paralyzing the diaphragm muscle (DIAm). There is gradual recovery of rhythmic DIAm activity ipsilateral to injury suggesting strengthened spared synaptic inputs to PMN. Neurotrophins play an important role in regulating synaptic function. Thus, we hypothesized that intrathecal brain‐derived neurotrophic factor (BDNF) enhances recovery of rhythmic phrenic activity after C2 spinal cord hemisection (C2HS), whereas quenching neurotrophins with TrkB‐IgG impairs functional recovery. An intrathecal catheter was placed at C4 to chronically infuse artificial CSF, BDNF or TrkB‐Fc using an osmotic pump. DIAm EMG electrodes were implanted bilaterally to verify complete hemisection at the time of C2HS and 3D post‐C2HS. In the artificial CSF group, functional recovery was evident in 33% of C2HS rats at 7D and 56% of rats at 14D post‐injury. Intrathecal BDNF treatment enhanced functional recovery, with 88% of rats at 7D and 100% at 14D post‐C2HS displaying rhythmic phrenic activity. In contrast, intrathecal TrkB‐Fc treatment completely prevented functional recovery up to 14D post‐C2HS. Our results demonstrate that BDNF/TrkB signaling at the level of the PMN pool is both sufficient and necessary for functional recovery of rhythmic phrenic activity after C2HS. Supported by NIH grant AR51173.

Myasthenia gravis is a disease of the neuromuscular junction (NMJ) associated with autoimmune destruction of the motor end‐plate. Inhibitors of acetylcholine (ACh) esterase are commonly used to mitigate symptoms by increasing ACh availability at the NMJ. Brain‐derived neurotrophic factor (BDNF) improves neuromuscular transmission in the rat diaphragm muscle (DIAm) by maintaining ACh release with repeated activation. We hypothesized that BDNF will mitigate the deficit of neuromuscular transmission in a passive transfer model of myasthenia gravis. Female Lewis rats were injected with vehicle, McAb1 (that does not induce myasthenia), and a myasthenogenic McAb3 antibody. Neuromuscular transmission failure (NMTF) was determined in DIAm‐phrenic nerve preparations by comparing the isometric force evoked by phrenic nerve stimulation and that evoked by superimposed direct muscle stimulation. After 2 min of repetitive stimulation, neuromuscular transmission was significantly impaired in myasthenic animals (NMTF: 80%) compared to vehicle and McAb1 animals (NMTF: ~60%). Exogenous BDNF treatment significantly improved neuromuscular transmission in all groups and NMTF in McAb3‐treated rats was no longer different from control or McAb1‐treated animals. We conclude that BDNF improves neuromuscular transmission in adult myasthenic rats. Supported by a Career Development Award from Mayo Clinic.

Neuregulin (NRG) is important for expression of acetylcholine (ACh) receptors during development of the neuromuscular junction (NMJ). NRG treatment significantly increases ACh‐induced currents in hippocampal neurons, but it is not known if NRG exerts a similar effect at NMJs. Diaphragm muscle (DIAm)‐phrenic nerve preparations from 7‐ and 14‐day old rats were used to determine the effects of NRG (heregulin β177–244) on neuromuscular transmission and DIAm contractile properties. Neuromuscular transmission failure (NMTF) was assessed by the difference in force elicited by repetitive phrenic nerve stimulation (0.2 ms pulses in 40 Hz, 330 ms trains) and superimposed, intermittent direct DIAm stimulation (1 ms pulses in 40 Hz, 330 ms trains applied every 15 seconds). NMTF was higher in 7‐day old compared to 14‐day old rats (76% vs. 65%; p<0.05). Exogenous NRG treatment significantly improved neuromuscular transmission in 7‐day old rats (NMTF: 52%; p<0.05), but not in 14‐day old rats (65%). Isometric DIAm contractile properties were not affected by NRG treatment in either age group. These results indicate that NRG has important synaptic effects during early postnatal development of the DIAm. Whether NRG effects on neuromuscular transmission result from acute changes in AChR expression or alternative mechanisms remains to be explored. Supported by NIH grant AR51173 and a Career Development Award from Mayo Clinic.

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.