Spinal synaptic enhancement with acute intermittent hypoxia improves respiratory function after chronic cervical spinal cord injury

Abstract

Respiratory insufficiency is the leading cause of death after high-cervical spinal cord injuries (SCIs). Although respiratory motor recovery can occur with time after injury, the magnitude of spontaneous recovery is limited. We hypothesized that partial respiratory motor recovery after chronic cervical SCI could be strengthened using a known stimulus for spinal synaptic enhancement, intermittent hypoxia. Phrenic motor output was recorded before and after intermittent hypoxia from anesthetized, vagotomized, and pump-ventilated control and C2 spinally hemisected rats at 2, 4, and 8 weeks after injury. Weak spontaneous phrenic motor recovery was present in all C2-injured rats via crossed spinal synaptic pathways that convey bulbospinal inspiratory premotor drive to phrenic motoneurons on the side of injury. Intermittent hypoxia augmented crossed spinal synaptic pathways [phrenic long-term facilitation; pLTF] for up to 60 min after hypoxia at 8 weeks, but not 2 weeks, after injury. Ketanserin, a serotonin 2A receptor antagonist, administered before intermittent hypoxia at 8 weeks after injury prevented pLTF. Serotonergic innervation near phrenic motoneurons was assessed after injury. The limited magnitude of pLTF at 2 weeks was associated with an injury-induced reduction in serotonin-containing nerve terminals in the vicinity of phrenic motoneurons ipsilateral to C2 hemisection. Thereafter, pLTF magnitude progressively increased with the recovery of serotonergic innervation in the phrenic motor nucleus. Intermittent hypoxia (or pLTF) has intriguing possibilities as a therapeutic tool, because its greatest efficacy may be in patients with chronic SCI, a time when most patients have already achieved maximal spontaneous functional recovery.

Figures

Figure 1.

Representative phrenic neurograms on the side of injury from control and spinally injured rats at 2 and 8 weeks after injury during baseline conditions, hypoxia, and 60 min after hypoxia (LTF). Spinally injured rats show partial recovery of baseline phrenic burst amplitude (volts) that increases progressively with time after injury (note different y-axis scales). Phrenic burst frequency also progressively increases after SCI. All rats show a robust phrenic amplitude response to hypoxia. pLTF is absent at 2 weeks but recovers by 8 weeks after injury.

Figure 2.

Inspiratory phrenic nerve activity during baseline, hypoxic, and posthypoxic (pLTF) conditions in control (CTL) and spinally injured rats at 2 weeks (2-W), 4 weeks (4-W), and 8 weeks (8-W) after injury. SCI decreased baseline moving-averaged (∫) phrenic burst amplitude and amplitude indexed to hypercapnic maximal amplitude (%MAX). Hemisection also decreased the change in moving-averaged (∫) phrenic burst amplitude as absolute values but not when indexed to hypercapnic maximal amplitude (%MAX). Finally, SCI minimized crossed spinal pLTF at 2 weeks after injury; pLTF recovered by 8 weeks after injury. pLTF was larger in LW versus SD rats. *, Different from controls; †, different from 2 weeks after injury; b, different from SD rats.

Figure 3.

Serotonin immunoreactivity (5HT-ir) correlates with phrenic LTF on the side of injury in control (CTL) and spinally injured (C2HS) rats at 2 weeks (2-W), 4 weeks (4-W), and 8 weeks (8-W) after injury. Ketanserin (KTSRN), a 5-HT2A receptor antagonist, abolished LTF at 8 weeks after injury. The magnitude of pLTF on the side of injury was positively correlated to the number of pixels occupied by serotonin-positive terminals in the C4 ventral horn (p < 0.05). *, Different from controls; †, different from 2 weeks after injury; ‡, different from 8 weeks after injury.

Figure 4.

Photomicrographs of and recovery in serotonergic innervation near labeled phrenic motoneurons in control (CTL) and spinally injured rats at 2 weeks (2-W) and 8 weeks (8-W) after injury. Phrenic motoneurons (red) were back-labeled with Dextran conjugated to Texas Red and visualized with fluorescent microscopy. *, Different from controls; †, different from 2 weeks after injury.