Protocol-Specific Effects of Intermittent Hypoxia Pre-Conditioning on Phrenic Motor Plasticity in Rats with Chronic Cervical Spinal Cord Injury

Abstract

"Low-dose" acute intermittent hypoxia (AIH; 3-15 episodes/day) is emerging as a promising therapeutic strategy to improve motor function after incomplete cervical spinal cord injury (cSCI). Conversely, chronic "high-dose" intermittent hypoxia (CIH; > 80-100 episodes/day) elicits multi-system pathology and is a hallmark of sleep apnea, a condition highly prevalent in individuals with cSCI. Whereas daily AIH (dAIH) enhances phrenic motor plasticity in intact rats, it is abolished by CIH. However, there have been no direct comparisons of prolonged dAIH versus CIH on phrenic motor outcomes after chronic cSCI. Thus, phrenic nerve activity and AIH-induced phrenic long-term facilitation (pLTF) were assessed in anesthetized rats. Experimental groups included: 1) intact rats exposed to 28 days of normoxia (Nx28; 21% O₂; 8 h/day), and three groups with chronic C2 hemisection (C2Hx) exposed to either: 2) Nx28; 3) dAIH (dAIH28; 10, 5-min episodes of 10.5% O₂/day; 5-min intervals); or 4) CIH (IH28-2/2; 2-min episodes; 2-min intervals; 8 h/day). Baseline ipsilateral phrenic nerve activity was reduced in injured versus intact rats but unaffected by dAIH28 or IH28-2/2. There were no group differences in contralateral phrenic activity. pLTF was enhanced bilaterally by dAIH28 versus Nx28 but unaffected by IH28-2/2. Whereas dAIH28 enhanced pLTF after cSCI, it did not improve baseline phrenic output. In contrast, unlike shorter protocols in intact rats, CIH28-2/2 did not abolish pLTF in chronic C2Hx. Mechanisms of differential responses to dAIH versus CIH are not yet known, particularly in the context of cSCI. Further, it remains unclear whether enhanced phrenic motor plasticity can improve breathing after cSCI.

Keywords: acute intermittent hypoxia; cervical spinal cord injury; chronic intermittent hypoxia; phrenic long-term facilitation; respiratory rehabilitation; sleep apnea.

FIG. 1.

C2 Hemi-section model and intermittent hypoxia protocols. (A) Schema depicting C2 spinal cord hemi-section injury model. Briefly, the left side of the spinal cord was cut at C2, severing descending projections to ipsilateral phrenic motoneurons, paralyzing the ipsilateral hemidiaphragm. (B) Schema depicting 28 days of normoxia (Nx28), daily acute intermittent hypoxia (dAIH28), and intermittent hypoxia simulating moderate sleep apnea (IH28-2/2) pre-conditioning protocols. Pre-conditioning was conducted daily for 4 weeks, beginning 8 weeks post-injury (or the equivalent timepoint in intact rats). Nx28 consisted of 8 h of continuous normoxia (21% O₂) per day. dAIH consisted of ten 5-min episodes of 10.5% O₂, with 5-min normoxic intervals. IH28-2/2 consisted of 8 h of 2-min hypoxic episodes (10.5% O₂), with 2-min normoxic intervals.

FIG. 2.

Left phrenic nerve activity (ipsilateral to C2 hemisection) during baseline and chemoreflex stimulated conditions. (A) Representative compressed traces depict raw (bottom) and integrated (top) left phrenic (i.e., ipsilateral to injury) neurograms under baseline conditions from intact rats, and rats with chronic C2 hemisection (C2Hx), exposed to 28 days of normoxia (Nx28), daily acute intermittent hypoxia (dAIH28), or chronic intermittent hypoxia simulating moderate sleep apnea (IH28-2/2). (B) Under baseline conditions, average ipsilateral/left integrated phrenic burst amplitude (volts) was reduced in all C2Hx rats vs. intact rats exposed to Nx28. In injured rat groups, no protocol-induced differences could be detected. (C) Representative traces depict raw and integrated left phrenic neurograms under chemoreflex stimulated conditions. (D) During chemoreflex stimulation, average ipsilateral/left integrated phrenic burst amplitude was reduced in C2Hx vs. intact rats. No IH protocol-specific differences were observed in injured rats. *Significantly different from intact Nx28; p < 0.05.

FIG. 3.

Right phrenic nerve activity (contralateral to C2 hemisection) during baseline and chemoreflex stimulated conditions. (A) Representative compressed traces depict raw (bottom) and integrated (top) right phrenic (i.e., contralateral to injury) neurograms under baseline conditions from intact rats, and rats with chronic C2 hemisection (C2Hx) exposed to 28 days of normoxia (Nx28), daily acute intermittent hypoxia (dAIH28), or chronic intermittent hypoxia simulating moderate sleep apnea (IH28-2/2). (B) Under baseline conditions, average contralateral/right integrated phrenic burst amplitude (volts) in C2Hx rats was not different from intact rats exposed to Nx28. No protocol-specific differences were observed across groups. (C) Representative traces depict raw and integrated right phrenic neurograms under hypercapnic chemoreflex stimulated conditions. (D) During chemoreflex stimulation, average contralateral/right integrated phrenic burst amplitude was not different between intact vs. injured rats. No protocol-specific differences were observed across groups.

FIG. 4.

Baseline phrenic nerve burst frequency. Average phrenic nerve burst frequency during baseline conditions. Baseline frequency was increased following C2 hemisection (C2Hx); no intermittent hypoxia (IH) protocol-specific differences were observed. *Significantly different from intact 28 days of normoxia (Nx28), p < 0.05.

FIG. 5.

Short-term hypoxic phrenic responses. (A) Average ipsilateral integrated phrenic nerve burst amplitude (volts) during the first (H1), second (H2), and third (H3) hypoxic episodes in each rat. Average phrenic nerve amplitude was reduced ipsilateral to injury during hypoxic episodes vs. intact rats, despite similar levels of arterial PaO₂. No intermittent hypoxia (IH) protocol-specific differences were observed, nor was there evidence for progressive augmentation in any group. (B) Average contralateral integrated phrenic nerve burst amplitude during the first, second, and third hypoxic episodes. Average phrenic nerve amplitude was not different between groups, nor was there evidence for progressive augmentation in successive hypoxic episodes. *Significantly different from intact 28 days of normoxia (Nx28); p < 0.05.

FIG. 6.

Phrenic long-term facilitation (pLTF) following moderate acute intermittent hypoxia (mAIH). (A) Representative traces depict integrated ipsilateral (left) phrenic neurograms at baseline (black) overlaid on neurogram traces 90 min post-hypoxia (red). (B) Average ipsilateral pLTF (% increase from baseline) was enhanced in rats with chronic C2 hemisection (C2Hx) exposed to daily acute intermittent hypoxia (dAIH28) vs. intact rats. In C2Hx rats exposed to intermittent hypoxia simulating moderate sleep apnea (IH28-2/2), pLTF was not different from intact 28 days of normoxia (Nx28) rats; an apparent difference from dAIH28 rats did not reach statistical significance (p = 0.168). (C) Representative traces depict integrated contralateral (right) phrenic neurograms at baseline (gray) vs. 90 min post-mAIH (black). (D) Average contralateral pLTF was enhanced in dAIH-treated rats with chronic C2Hx. Although C2Hx rats exposed to IH28-2/2 tended to have blunted pLTF (vs. dAIH-treated), this difference did not reach statistical significance (p = 0.1). *Significantly different from intact Nx28; p < 0.05. #Significantly different from C2Hx Nx28; p < 0.05.

FIG. 7.

Mean arterial pressure and heart rate. (A) Representative traces depict instantaneous heart rate (top) and arterial blood pressure (bottom) under baseline conditions. (B) Average baseline mean arterial pressure (mm Hg) was not different between groups. (C) Average baseline heart rate (beats/min) was not different between groups.