Zhan, Wen‐Zhi

Muscle protein content reflects a balance between protein synthesis and degradation, which are likely regulated by neuromuscular activity and/or neurotrophin influence. In previous studies, we found that unilateral denervation (DNV) of the rat diaphragm muscle (DIAm) decreases myosin heavy chain (MHC) protein content after approximately 7 days. However, we did not determine whether the decrease in DIAm MHC protein content was the result of a decrease in protein synthesis, an increase in protein degradation, or both. We hypothesize that protein degradation plays a significant role in the decrease in DIAm protein content following DNV. In this study, we analyzed the ubiquitination state of cellular proteins as a marker of protein degradation. Conjugation of ubiquitin to cellular proteins targets proteins for degradation by proteosomes. We analyzed DIAm from adult male Sprague‐Dawley rats 3, 7 and 14 days after DNV. The DIAm was homogenized and protein ubiquitination was analyzed by Western analysis. No difference in ubiquitination was detected between control (CTL), sham surgery‐treated (sham), and 3‐day DNV animals. However, following 7 and 14 days of DNV, there was an approximate five‐fold increase in protein ubiquitination in DIAm compared to CTL and sham groups. These results demonstrate that DIAm protein ubiquitination increases between 3 and 7 days following DNV, and support the hypothesis that protein degradation contributes to the DNV‐induced decrease in DIAm MHC protein content. Supported by NIH grants AR51173 and HL37680.

Following spinal cord hemisection at C2 (SH), removal of descending excitatory drive to phrenic motoneurons results in ipsilateral diaphragm muscle paralysis with partial spontaneous recovery of rhythmic phrenic activity over time. Brain‐derived neurotrophic factor (BDNF) plays an important role in neuroplasticity following SH. Intraspinal transplantation of mesenchymal stem cells engineered to produce BDNF‐GFP (BDNF‐MSCs) at the time of injury promotes functional recovery of all animals, despite low levels of MSC survival at 14 days post‐SH. The goal of the present study was to determine the survival and localization of BDNF‐MSCs after intraspinal transplantation. BDNF‐MSCs were injected intraspinally at C4 (2 sites, total 2 x 10 5 cells in 4 µl) in adult male Sprague‐Dawley rats. After one and three days, the cervical spinal cord was fixed, excised, and cut longitudinally. Survival of transplanted BDNF‐MSCs was confirmed in all animals at 1 day post‐transplantation (n=5) and in five of 6 animals at 3 days post‐transplantation. BDNF‐MSCs remained localized at the site of injection in the white matter at 1 and 3 days, with an average volume of 1.1 ± 0.3 µl. There was no localization of MSCs in the vicinity of phrenic motoneurons identified by retrograde labeling with cholera toxin subunit B. Understanding the time course of stem cell survival after transplantation is important in defining windows of opportunity during which the beneficial effects of BDNF on respiratory function after spinal cord injury are executed. Supported by HL96750 and Mayo Clinic Center for Regenerative Medicine.

Upper cervical spinal cord injury disrupts descending inspiratory‐related drive to phrenic motoneurons (PMNs) paralyzing the diaphragm muscle (DIAm). There is gradual recovery of rhythmic DIAm activity ipsilateral to injury suggesting strengthened spared synaptic inputs to PMNs. We previously showed that intrathecal treatment with brain‐derived neurotrophic factor (BDNF) enhances recovery of rhythmic DIAm activity after C2 spinal cord hemisection (C2SH). We hypothesized that TrkB signaling in PMN is necessary for functional recovery post‐C2SH. Using a recently validated intrapleural technique, daily injections of TrkB siRNA were administered to adult male Sprague‐Dawley rats. Bilateral DIAm EMG activity was chronically recorded before and after C2SH to assess recovery of rhythmic DIAm activity. In the vehicle‐treated group, functional recovery was evident in ~10% of C2SH rats at 7D and ~40% of rats at 14D post‐injury. In contrast, none of the TrkB siRNA treated rats displayed functional recovery up to 14D post‐C2SH. Transport of intrapleural Cy3‐labeled siRNA was verified in retrogradely‐labeled phrenic motoneurons. Our results indicate that BDNF/TrkB signaling at PMNs is necessary for functional recovery of rhythmic DIAm activity after C2SH. Supported by NIH grant R01 HL096750 and Mayo Clinic

Following spinal cord hemisection at C2 (C2SH), removal of descending excitatory drive to phrenic motoneurons results in ipsilateral diaphragm muscle (DIAm) paralysis. Over time, there is partial recovery of rhythmic phrenic activity post‐C2SH. Intrathecal delivery of brain‐derived neurotrophic factor (BDNF) enhances this functional recovery, with 100% of rats displaying rhythmic phrenic activity at 14 days (14D) post‐C2SH; but in humans intrathecal BDNF is associated with adverse effects that limit application. Stem cell transplantation may facilitate a targeted BDNF delivery and minimize side effects. In adult rats, bilateral DIAm EMG activity was chronically monitored before and after C2SH to assess recovery. Wild type, GFP‐expressing bone‐marrow derived mesenchymal stem cells (MSC‐wt) or BDNF‐GFP expressing MSC (MSC‐BDNF) were injected at C2. Seventy % of MSC‐BDNF treated rats displayed functional recovery at 7D and 100% at 14D post‐C2SH. Functional recovery in BDNF‐wt treated C2SH rats was not different from untreated C2SH controls (~10% at 7D and ~40% at 14D). Cellular incorporation of MSC into the spinal cord was evident by GFP immunoreactivity in both MSC groups. We conclude that targeted MSC‐based delivery of BDNF enhances functional recovery of DIAm respiratory activity following an upper cervical spinal cord injury. Supported by NIH grant HL096750 and Mayo Clinic.

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.