Repetitive intermittent hypoxia induces respiratory and somatic motor recovery after chronic cervical spinal injury

Abstract

Spinal injury disrupts connections between the brain and spinal cord, causing life-long paralysis. Most spinal injuries are incomplete, leaving spared neural pathways to motor neurons that initiate and coordinate movement. One therapeutic strategy to induce functional motor recovery is to harness plasticity in these spared neural pathways. Chronic intermittent hypoxia (CIH) (72 episodes per night, 7 nights) increases synaptic strength in crossed spinal synaptic pathways to phrenic motoneurons below a C2 spinal hemisection. However, CIH also causes morbidity (e.g., high blood pressure, hippocampal apoptosis), rendering it unsuitable as a therapeutic approach to chronic spinal injury. Less severe protocols of repetitive acute intermittent hypoxia may elicit plasticity without associated morbidity. Here we demonstrate that daily acute intermittent hypoxia (dAIH; 10 episodes per day, 7 d) induces motor plasticity in respiratory and nonrespiratory motor behaviors without evidence for associated morbidity. dAIH induces plasticity in spared, spinal pathways to respiratory and nonrespiratory motor neurons, improving respiratory and nonrespiratory (forelimb) motor function in rats with chronic cervical injuries. Functional improvements were persistent and were mirrored by neurochemical changes in proteins that contribute to respiratory motor plasticity after intermittent hypoxia (BDNF and TrkB) within both respiratory and nonrespiratory motor nuclei. Collectively, these studies demonstrate that repetitive acute intermittent hypoxia may be an effective and non-invasive means of improving function in multiple motor systems after chronic spinal injury.

Figures

Figure 1.

dAIH restores ventilatory capacity in rats with cervical (C2) spinal hemisection. Using whole-body plethysmography (d), V̇E (a) and its components, VT (b) and breathing frequency (c), were assessed during air breathing (baseline) and when breathing an hypercapnic/hypoxic gas mixture (7% CO2/10.5% O2) to assess ventilatory capacity. Neither C2HS (C2HS-norm; gray bars) nor dAIH in hemisected rats (C2HS-dAIH; black bars) affected V̇E during baseline conditions. However, C2HS decreased ventilatory capacity during maximal chemoreceptor stimulation versus sham rats (p = 0.04). dAIH increased ventilatory capacity after C2HS, restoring ventilation during maximal chemoreceptor stimulation to normal levels (not significantly different vs sham rats). C2HS decreased VT during baseline (25%; p = 0.036) and chemoreceptor-stimulated breathing (22%; p = 0.005); dAIH restored 70% of this lost capacity to increase VT during chemoreceptor stimulation in C2HS rats (p = 0.028). Breathing frequency was increased by C2HS during baseline conditions (29%; p < 0.001), although not during chemoreceptor stimulation; frequency was not affected by dAIH in either condition. *p < 0.05 versus sham animals; †p < 0.05 versus C2HS-dAIH.

Figure 2.

dAIH strengthens spontaneous phrenic nerve activity and crossed-spinal synaptic pathways to phrenic motor neurons after C2HS. In a and b, integrated phrenic neurograms are shown to illustrate spontaneous phrenic nerve activity before and after dAIH in rats with C2HS. Whereas dAIH increased spontaneous phrenic activity ipsilateral to hemisection during baseline and maximal chemoreceptor stimulation (a), no effect was observed on the side contralateral to injury (b). In c, dAIH effects on spontaneous phrenic nerve activity on the injured side are summarized; data are expressed as a percentage of the uninjured side during baseline conditions. At each level of chemoreceptor stimulation (baseline, increases in arterial PCO2 of 20 and 40 mmHg above baseline, and 40 mmHg PCO2 increase plus hypoxia), dAIH increased the amplitude of integrated phrenic bursts (filled bars) versus normoxia-treated hemisected rats (open bars), demonstrating induced functional recovery. In d, the amplitude of phrenic responses evoked by electrical stimulation of the contralateral ventrolateral funiculus are shown. The amplitude of evoked phrenic responses is greater in dAIH-treated (filled bars) versus normoxia-treated (open bars) rats at all stimulus intensities. *p < 0.05 versus sham rats.

Figure 3.

Forelimb ladder-stepping ability improves after dAIH in rats with incomplete cervical spinal injuries. In a, forelimb ladder-stepping performance was assessed in spinally injured rats with and without dAIH treatment. In b, rats exposed to dAIH beginning 4 weeks after transection of the dorsolateral spinal funiculus at C3 (filled circles; n = 7) made significantly fewer errors with the impaired forelimb beginning at day 4 of treatment versus injured rats exposed to normoxia only (sham, open circles, n = 7). Treatment days are indicated by arrows. Full functional recovery lasted ≥3 weeks after dAIH treatment. Performance was measured as the number of correct steps/total steps while walking over a horizontal ladder; measurements were made before surgery (Pre-Sx), 4 weeks after surgery but before dAIH (Pre-AIH), 1 h after AIH on each treatment day (arrows), and at 7 and 21 d after the AIH treatments had ended. *p < 0.05 versus sham rats at the same time point.

Figure 4.

dAIH and C2HS upregulate BDNF, TrkB, and phospho-TrkB in C4 and C7 motor nuclei. In a–c, examples of BDNF, TrkB, and phosphorylated TrkB (pTrkB) immunoreactivity are shown in the region of the phrenic motor nucleus (C4) from rats with dAIH plus C2HS (C2HS + dAIH). Other conditions are not shown because of space limitations. Boxes indicate the region of the phrenic motor nucleus in which densitometry was performed; higher-magnification images from this region are in the bottom right corner of these images. Scale bars: lower magnification, 200 μm; higher magnification, 50 μm (same scale in all panels). In d–f, summaries of densitometry at C4 are provided for each protein in rats from the following groups (each n = 5): (1) sham-operated rats exposed to normoxia, (2) sham-operated rats treated with dAIH, (3) rats 2 weeks after C2HS exposed to normoxia, and (4) rats 2 weeks after C2HS exposed to dAIH. Although both dAIH and C2HS tended to increase BDNF, their combined effect was not greater than either treatment alone. TrkB and phospho-TrkB were increased by dAIH and C2HS, and their combined effect was greater than either treatment alone (particularly pTrkB). Greater TrkB phosphorylation suggests activation of signaling pathways that underlie phrenic motor plasticity. In g–i, examples of BDNF, TrkB, and phosphorylated TrkB immunoreactivity are shown in the C7 ventral horn from rats with dAIH plus C2HS (C2HS + dAIH). Scale bar: 200 μm. In j–l, summaries of densitometry at C7 are provided for the following groups: (1) sham-operated rats exposed to normoxia, (2) sham-operated rats treated with dAIH, (3) rats 2 weeks after C2HS exposed to normoxia, and (4) rats 2 weeks after C2HS exposed to dAIH. Densitometry was performed in spinal lamina 9. Results from C7 are strikingly similar to C4, indicating that dAIH and C2HS elicit similar effects in spinal respiratory and somatic motor nuclei. Data are means ± 1 SEM. *p < 0.05 versus normoxia; †p < 0.05 versus dAIH; ‡p < 0.05 versus C2HS.

Figure 5.

dAIH does not induce hippocampal gliosis or neuron death. Because CIH induces CNS inflammation and hippocampal cell death, we determined whether the more modest (dAIH) protocol elicits similar adverse effects. There was no evidence for reactive astrocytes (GFAP positive; a–c) or microglia (OX-42 positive; d–f) after dAIH. Cell counts reveal that the density of GFAP-positive (c) and OX-42 positive (f) cells was unaffected by dAIH (n = 5 per group), and there were no obvious increases in the intensity of either cell marker (a vs b; d vs e). Furthermore, there was no evidence for dAIH-induced hippocampal apoptosis (g, h) or nonspecific cell death (i, j) based on TUNEL or cresyl violet staining, respectively. h′ is a positive control for TUNEL staining taken from the cortex after middle cerebral artery occlusion in a rat. Magnified images of the CA2 hippocampal subfield from control and dAIH-treated rats are presented in the bottom right corners of g and h. Some sections were stained with cresyl violet to enable morphological assessment (i, j). Data in c and f are means ± 1 SEM. Scale bars: a, b, d, e, 50 μm; g, h, 500 μm; i, j, 100 μm.