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.

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.

Intermittent hypoxia elicits protocol-dependent effects on hypoglossal (XII) motor plasticity. Whereas low-dose, acute intermittent hypoxia (AIH) elicits serotonin-dependent plasticity in XII motor neurons, high-dose, chronic intermittent hypoxia (CIH) elicits neuroinflammation that undermines AIH-induced plasticity. Preconditioning with repeated AIH and mild CIH enhance AIH-induced XII motor plasticity. Since intermittent hypoxia pre-conditioning could enhance serotonin-dependent XII motor plasticity by increasing serotonergic innervation density of the XII motor nuclei, we tested the hypothesis that 3 distinct intermittent hypoxia protocols commonly studied to elicit plasticity (AIH) or simulate aspects of sleep apnea (CIH) differentially affect XII serotonergic innervation. Sleep apnea and associated CIH are common in people with cervical spinal injuries and, since repetitive AIH is emerging as a promising therapeutic strategy to improve respiratory and non-respiratory motor function after spinal injury, we also tested the hypotheses that XII serotonergic innervation is increased by repetitive AIH and/or CIH in rats with cervical C2 hemisections (C2Hx). Serotonergic innervation was assessed via immunofluorescence in male Sprague Dawley rats, with and without C2Hx (beginning 8 weeks post-injury) exposed to 28 days of: 1) normoxia; 2) daily AIH (10, 5-min 10.5% O2 episodes per day; 5-min normoxic intervals); 3) mild CIH (5-min 10.5% O2 episodes; 5-min intervals; 8 h/day); and 4) moderate CIH (2-min 10.5% O2 episodes; 2-min intervals; 8 h/day). Daily AIH, but neither CIH protocol, increased the area of serotonergic immunolabeling in the XII motor nuclei in both intact and injured rats. C2Hx per se had no effect on XII serotonergic innervation density. Thus, daily AIH may increases XII serotonergic innervation and function, enhancing the capacity for serotonin-dependent, AIH-induced plasticity in upper airway motor neurons. Such effects may preserve upper airway patency and/or swallowing ability in people with cervical spinal cord injuries and other clinical disorders that compromise breathing and airway defense.