Respiratory Axon Plasticity Drives Recovery of Diaphragm Function After Spinal Cord Injury

We are testing a novel strategy to promote axonal growth of damaged descending bulbospinal respiratory axons and reinnervate phrenic motor neurons (PhMN) to restore diaphragm function after cervical spinal cord injury (SCI) in rats. SCI is caused by trauma to the spinal cord, and more than half of all cases occur in the cervical region, leading to breathing compromise by damaging circuits involved in respiratory control. Restoration of functional deficits caused by SCI is limited due to cell-intrinsic and -extrinsic barriers to axon plasticity and a lack of guidance cues to signal growing axons to appropriate targets. The C3-C5 mid-cervical spinal cord levels house the PhMNs, which are responsible for diaphragm activation. PhMNs are predominately mono-synaptically innervated by supraspinal respiratory neurons located in a brainstem nucleus called the rostral Ventral Respiratory Group (rVRG). We are seeking to reverse respiratory dysfunction after SCI by restoring the crucial circuit controlling PhMNs, and thus diaphragm activation. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family of growth factors, promotes axonal growth and acts as a guidance cue. We aim to promote targeted reinnervation of PhMNs and restore diaphragm function by overexpressing BDNF focally at the location of denervated PhMNs via an adeno-associated virus (AAV) to direct growing axons. In Part 1, we determined whether providing a focal source of the axon guidance molecule, BDNF, promoted diaphragmatic recovery after cervical SCI. We assessed for rVRG-PhMN reconnection by measuring restoration of diaphragm function by testing in vivo diaphragm activation via electromyography (EMG). We found that the EMG diaphragm amplitude was significantly increased in AAV2-BDNF treated rats compared to AAV2-GFP controls eight weeks after C2 hemisection, indicating restoration of diaphragm activation. In Part 2, we determined whether focal BDNF upregulation promoted rVRG-PhMN circuit re-connectivity and targeted PhMN reinnervation by rVRG axons following cervical SCI. We assessed rVRG axons using an AAV vector expressing an anterograde tracer, examining regrowth and collateral sprouting, and identified synaptic reconnection with spared PhMNs by markers of putative synaptic connections. Excitingly, we used this labeling technique to distinguish between modes of recovery, i.e. ipsilateral regrowth versus contralateral sprouting by selectively labeling subpopulations of rVRG axons. We found that focal BDNF upregulation promoted increased synaptic connections on denervated PhMNs from spared axons originating in the contralateral rVRG and from serotonergic axons. We aimed to use the strategy proposed here to reconnect motor neurons responsible for diaphragm activation with respiratory centers in the medulla to restore respiratory function following disruption after cervical SCI. The potential therapeutic benefits being explored will have profound implications for SCI patients suffering respiratory dysfunction