Abstract
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.
Highlights
Most spinal cord injuries (SCI) are incomplete, with some sparing of spinal cord pathways
Our results demonstrate that the intrapleural delivery technique [28] combined with the selective transduction of phrenic motoneurons by AAV7 provides a novel method of targeting gene expression to phrenic motoneurons, thereby promoting recovery of ipsilateral diaphragm EMG activity after Spinal Cord Hemisection (SH)
A total of 98 GFP immunoreactive phrenic motoneurons were identified out of 884 motoneurons labeled by cholera toxin subunit B (CTB) in six rats treated with intrapleural AAV7-GFP
Summary
Most spinal cord injuries (SCI) are incomplete, with some sparing of spinal cord pathways. Following SH, there is spontaneous recovery of ipsilateral rhythmic phrenic activity due to strengthening of latent contralateral excitatory premotor input to phrenic motoneurons [9,10,11,12,13,14]. This spontaneous neuroplasticity results in recovery of some ipsilateral diaphragm function after SH, significant impairments remain [7,10,15]. Expression of serotonergic and glutamatergic receptors within phrenic motoneurons increases following SH [6,24], and the time course of changes generally corresponds with onset of spontaneous recovery of rhythmic phrenic activity. Our results demonstrate that the intrapleural delivery technique [28] combined with the selective transduction of phrenic motoneurons by AAV7 provides a novel method of targeting gene expression to phrenic motoneurons, thereby promoting recovery of ipsilateral diaphragm EMG activity after SH
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