Sex differences in spontaneous respiratory recovery following chronic C2 hemisection

Abstract

Respiratory deficits after C2 hemisection (C2Hx) have been well documented through single-sex investigations. Although ovarian sex hormones enable enhanced respiratory recovery observed in females 2 wk post-C2Hx, it remains unknown if sex impacts spontaneous respiratory recovery at chronic time points. We conducted a longitudinal study to provide a comprehensive sex-based characterization of respiratory neuromuscular recovery for 8 wk after C2Hx. We recorded ventilation and chronic diaphragm electromyography (EMG) output in awake, behaving animals, phrenic motor output in anesthetized animals, and performed diaphragm muscle histology in chronically injured male and female rodents. Our results show that females expressed a greater recovery of tidal volume and minute ventilation compared with males during subacute and chronic time points. Eupneic diaphragm EMG amplitude during wakefulness and phrenic motor amplitude are similar between sexes at all time points after injury. Our data also suggest that females have a greater reduction in ipsilateral diaphragm EMG amplitude during spontaneous deep breaths (e.g., sighs) compared with males. Finally, we show evidence for atrophy and remodeling of the fast, fatigable fibers ipsilateral to injury in females, but not in males. To our knowledge, the data presented here represent the first study to report sex-dependent differences in spontaneous respiratory recovery and diaphragm muscle morphology following chronic C2Hx. These data highlight the need to study both sexes to inform evidence-based therapeutic interventions in respiratory recovery after spinal cord injury (SCI).NEW & NOTEWORTHY In response to chronic C2 hemisection, female rodents display increased tidal volume during eupneic breathing compared with males. Females show a greater reduction in diaphragm electromyography (EMG) amplitude during spontaneous deep breaths (e.g., sighs) and atrophy and remodeling of fast, fatigable diaphragm fibers. Given that most rehabilitative interventions occur in the subacute to chronic stages of injury, these results highlight the importance of considering sex when developing and evaluating therapeutics after spinal cord injury.

Keywords: breathing; diaphragm muscle; respiratory recovery; sex differences; spinal cord injury.

Graphical abstract

Figure 1.

Experimental protocol. Male and female rats were acclimated to modified cages and whole body plethysmography (WBP) chambers for five consecutive days, 1.5 h/day/environment. Animals received a custom diaphragm EMG implant and a left C2 hemisection (C2Hx). Diaphragm EMG output and ventilation were recorded before (preinjury) and weekly after injury for 8 wk. At 9 wk postinjury, terminal neurophysiology was performed to record phrenic nerve output, and both diaphragm muscle and spinal cord tissue were harvested. Neurophys, neurophysiology.

Figure 2.

Tidal volume and minute ventilation are elevated in females following C2Hx. Representative compressed (A) and expanded (B) traces of airflow (mL/breath/100 g) from a male (black line) and female (gray line) rat during normoxia and respiratory challenge. C: average tidal volume (mL/breath/100 g), breathing rate (breaths/min), and minute ventilation volume (mL/min/100 g) for males (circles) and females (squares) during normoxia. D: average tidal volume (mL/breath/100 g), breathing rate (breaths/min), and minute ventilation volume (mL/min/100 g) for males (circles) and females (squares) during respiratory challenge. Male n = 8, female n = 8. Values are means (SD). All two-way RM ANOVAs, Šídák’s post hoc. #Significantly different from males. Shaded shapes: different from preinjury. P < 0.05. C2Hx, C2 hemisection.

Figure 3.

Diaphragm EMG amplitude is similar in males and females following chronic C2Hx. A: schematic of the tethered approach to record diaphragm EMG during wakefulness (created with BioRender.com). B: representative ipsilateral and contralateral raw and integrated (∫) diaphragm EMG traces before (preinjury) and 8 wk after injury. C: average breathing frequency (Hz) at each time point. Two-way RM ANOVAs, Šídák’s post hoc. Average ipsilateral and contralateral diaphragm EMG amplitude (% preinjury) for males (D) and females (E) before and for 8 wk after injury. One-way RM ANOVAs, Šídák’s post hoc. F: sex-based comparison of ipsilateral diaphragm EMG amplitude (% preinjury). Two-way RM ANOVAs, Šídák’s post hoc. G: average ipsilateral diaphragm EMG amplitude (% of preinjury sigh amplitude) throughout the chronic protocol. Two-way RM ANOVA, Šídák’s post hoc. Male n = 6, female n = 7. Values are means (SD). Shaded shapes/bars: different from preinjury. P < 0.05. amp, amplitude; contra dia, contralateral diaphragm; C2Hx, C2 hemisection; EMG, electromyography; ipsi, ipsilateral.

Figure 4.

Ipsilateral diaphragm EMG amplitude during sigh is decreased in females with chronic C2Hx. A: representative ipsilateral and contralateral raw and integrated (∫) diaphragm EMG traces during a sigh before (preinjury) and at 8 wk postinjury. B: average ipsilateral sigh diaphragm EMG amplitude (% preinjury) for males and females before (preinjury) and at 2, 4, and 8 wk after injury. Male n = 6, female n = 7. Values are means (SD). Two-way RM ANOVA, Šídák’s post hoc. Shaded shapes/bars: different from preinjury. P < 0.05. amp, amplitude; contra dia, contralateral diaphragm; C2Hx, C2 hemisection; EMG, electromyography; ipsi, ipsilateral.

Figure 5.

Phrenic motor output is similar in males and females following chronic C2Hx. A: compressed integrated ipsilateral and contralateral phrenic neurograms during baseline, progressive hypercapnic, hypoxic, and combined hypercapnic/hypoxic challenges. Average ipsilateral (B) and contralateral (C) phrenic amplitude (V) during baseline and respiratory challenges in male and female rodents. Normalized average ipsilateral (D) and contralateral (E) phrenic amplitude (% baseline) during baseline and respiratory challenges in male and female rodents. Male n = 9, female n = 9. Values are means (SD). All two-way RM ANOVAs, Šídák’s post hoc. Shaded shapes/bars: different from baseline. P < 0.05. amp, amplitude; contra dia, contralateral diaphragm; C2Hx, C2 hemisection; ipsi, ipsilateral.

Figure 6.

Sex differences in diaphragm type IIx/b fibers in chronic C2Hx animals. Representative photomicrographs of the midcostal diaphragm stained for myosin heavy chain isoforms MyHC₁ (pink), MyHC_2A (green), MyHC_2B (red), MyHC_2X (unlabeled, black), and fiber border laminin (blue) for male (A) and female (D) rodents. Image shown at ×10 magnification. Average fiber type distribution (%) for male (B) and female (E) rodents following chronic C2Hx. Average diaphragm muscle fiber cross-sectional area (µm²) for male (C) and female (F) rodents following chronic C2Hx. Sex-based comparison of average ipsilateral fiber type distribution (%; G) and diaphragm muscle fiber cross-sectional area (µm²; H). Sex-based comparison of average contralateral fiber type distribution (%; I) and diaphragm muscle fiber cross-sectional area (µm²; J). Male n = 4, female n = 4. Values are means (SD). Two-way ANOVA; Tukey’s post hoc. *Different from contralateral. #Different from males. P < 0.05. C2Hx, C2 hemisection.

Figure 7.

Tissue sparing at the lesion epicenter is similar between males and females. A: representative C2 cross-section at the lesion epicenter stained with cresyl violet. Reconstructed traces of the lesion epicenter for males (B) and females (C). D: average tissue sparing (% total cord). Male (n = 7), female (n = 7). Values are means (SD). Unpaired, two-tailed, t test. P > 0.05.