Phrenic motor neuron survival below cervical spinal cord hemisection.

Cervical spinal cord injury (cSCI) severs bulbospinal projections to respiratory motor neurons, paralyzing respiratory muscles below the injury. C2 spinal hemisection (C2Hx) is a model of cSCI often used to study spontaneous and induced plasticity and breathing recovery post-injury. One key assumption is that C2Hx dennervates motor neurons below the injury, but does not affect their survival. However, a recent study reported substantial bilateral motor neuron death caudal to C2Hx. Since phrenic motor neuron (PMN) death following C2Hx would have profound implications for therapeutic strategies designed to target spared neural circuits, we tested the hypothesis that C2Hx minimally impacts PMN survival. Using improved retrograde tracing methods, we observed no loss of PMNs at 2- or 8-weeks post-C2Hx. We also observed no injury-related differences in ChAT or NeuN immunolabeling within labelled PMNs. Although we found no evidence of PMN loss following C2Hx, we cannot rule out neuronal loss in other motor pools. These findings address an essential prerequisite for studies that utilize C2Hx as a model to explore strategies for inducing plasticity and/or regeneration within the phrenic motor system, as they provide important insights into the viability of phrenic motor neurons as therapeutic targets after high cervical injury.

Serotonergic innervation of respiratory motor nuclei after cervical spinal injury: Impact of intermittent hypoxia.

Although cervical spinal cord injury (cSCI) disrupts bulbo-spinal serotonergic projections, partial recovery of spinal serotonergic innervation below the injury site is observed after incomplete cSCI. Since serotonin contributes to functional recovery post-injury, treatments to restore or accelerate serotonergic reinnervation are of considerable interest. Intermittent hypoxia (IH) was reported to increase serotonin innervation near respiratory motor neurons in spinal intact rats, and to improve function after cSCI. Here, we tested the hypotheses that spontaneous serotonergic reinnervation of key respiratory (phrenic and intercostal) motor nuclei: 1) is partially restored 12 weeks post C2 hemisection (C2Hx); 2) is enhanced by IH; and 3) results from sprouting of spared crossed-spinal serotonergic projections below the site of injury. Serotonin was assessed via immunofluorescence in male Sprague Dawley rats with and without C2Hx (12 wks post-injury); individual groups were exposed to 28 days of: 1) normoxia; 2) daily acute IH (dAIH28: 10, 5 min 10.5% O2 episodes per day; 5 min normoxic intervals); 3) mild chronic IH (IH28-5/5: 5 min 10.5% O2 episodes; 5 min intervals; 8 h/day); or 4) moderate chronic IH (IH28-2/2: 2 min 10.5% O2 episodes; 2 min intervals; 8 h/day), simulating IH experienced during moderate sleep apnea. After C2Hx, the number of ipsilateral serotonergic structures was decreased in both motor nuclei, regardless of IH protocol. However, serotonergic structures were larger after C2Hx in both motor nuclei, and total serotonin immunolabeling area was increased in the phrenic motor nucleus but reduced in the intercostal motor nucleus. Both chronic IH protocols increased serotonin structure size and total area in the phrenic motor nuclei of uninjured rats, but had no detectable effects after C2Hx. Although the functional implications of fewer but larger serotonergic structures are unclear, we confirm that serotonergic reinnervation is substantial following injury, but IH does not affect the extent of reinnervation.