Diaphragm electromyographic activity following unilateral midcervical contusion injury in rats

Abstract

Contusion-type injuries to the spinal cord are characterized by tissue loss and disruption of spinal pathways. Midcervical spinal cord injuries impair the function of respiratory muscles and may contribute to significant respiratory complications. This study systematically assessed the impact of a 100-kDy unilateral C4 contusion injury on diaphragm muscle activity across a range of motor behaviors in rats. Chronic diaphragm electromyography (EMG) was recorded before injury and at 1 and 7 days postinjury (DPI). Histological analyses assessed the extent of perineuronal net formation, white-matter sparing, and phrenic motoneuron loss. At 7 DPI, ∼45% of phrenic motoneurons were lost ipsilaterally. Relative diaphragm root mean square (RMS) EMG activity increased bilaterally across a range of motor behaviors by 7 DPI. The increase in diaphragm RMS EMG activity was associated with an increase in neural drive (RMS value at 75 ms after the onset of diaphragm activity) and was more pronounced during higher force, nonventilatory motor behaviors. Animals in the contusion group displayed a transient decrease in respiratory rate and an increase in burst duration at 1 DPI. By 7 days, following midcervical contusion, there was significant perineuronal net formation and white-matter loss that spanned 1 mm around the injury epicenter. Taken together, these findings are consistent with increased recruitment of remaining motor units, including more fatigable, high-threshold motor units, during higher force, nonventilatory behaviors. Changes in diaphragm EMG activity following midcervical contusion injury reflect complex adaptations in neuromotor control that may increase the risk of motor-unit fatigue and compromise the ability to sustain higher force diaphragm efforts.

New & noteworthy: The present study shows that unilateral contusion injury at C4 results in substantial loss of phrenic motoneurons but increased diaphragm muscle activity across a range of ventilatory and higher force, nonventilatory behaviors. Measures of neural drive indicate increased descending input to phrenic motoneurons that was more pronounced during higher force, nonventilatory behaviors. These findings reveal novel, complex adaptations in neuromotor control following injury, suggestive of increased recruitment of more fatigable, high-threshold motor units.

Keywords: chronic EMG recordings; neuromotor control; respiratory muscles; spinal cord injury.

Fig. 1.

Extent of perineuronal net formation around injury epicenter at 7 days following a unilateral C4 contusion injury. A: representative 20 μm-thick transverse sections from an individual unilateral C4 contusion animal displaying Wisteria floribunda agglutinin (WFA) immunoreactivity (grayscale; black reflects greater immunoreactivity) in 5 sections (∼200 μm apart), spanning 0.4 mm rostral to 0.4 mm caudal to the injury epicenter. An outline of the spinal cord section and contralateral gray matter is presented for clarity. B: summary of integrated WFA immunoreactivity (fluorescence intensity), 2.4 mm around the injury epicenter in laminectomy (n = 4) and contusion (n = 4) groups. A significant increase in perineuronal net formation was evident at a distance 0.4 mm rostral to 0.6 mm caudal to the injury site (*P < 0.001). Original scale bar, 1 mm.

Fig. 2.

White-matter sparing around the injury epicenter at 7 days following a unilateral C4 contusion injury. A: summary of percent white matter spared, 1.6 mm around the injury epicenter in laminectomy (n = 4) and contusion (n = 4) groups. Evidence of significant white-matter compromise is present, 1 mm around the injury site (*P < 0.001). Spinal cord cross-section is a representative 20-μm-thick eriochrome cyanine stained section at the injury epicenter of a C4 contusion animal. Gray-shaded areas represent expected location of descending bulbospinal inputs to phrenic motoneurons in the ventromedial and lateral columns. B: summary of percent white matter spared in the ipsilateral-ventral columns, 1.6 mm around the injury epicenter in laminectomy (n = 4) and contusion (n = 4) groups (*P < 0.001, mixed linear model; post hoc Tukey-Kramer HSD at P < 0.05). The ipsilateral-ventral area quantified is highlighted in red in the spinal cord cross-section.

Fig. 3.

Phrenic motoneuron counts in laminectomy and unilateral C4 contusion animals at 7 days postinjury (DPI). A: summary of phrenic motoneuron counts in the ipsilateral (Ipsi) and contralateral (Contra) spinal cord in laminectomy (n = 5) and contusion (n = 6) groups. Animals in the contusion group had extensive and significant phrenic motoneuron loss at 7 DPI (*P < 0.001, mixed linear model; post hoc Tukey-Kramer HSD at P < 0.05). B: representative image of retrogradely labeled phrenic motoneurons obtained from consecutive maximum intensity projections of 100 μm-thick longitudinal spinal cord sections for an animal with a unilateral C4 contusion injury. *Artifact in fluorescent image. Original scale bar, 0.5 mm.

Fig. 4.

Representative ipsilateral and contralateral compound diaphragm EMG and root mean square (RMS) EMG recordings across eupnea, hypoxia-hypercapnia (10% O₂, 5% CO₂), airway occlusion, and spontaneous deep breath (sigh) from a C4 contusion animal at −1 and 7 days postinjury. Note increased diaphragm RMS EMG activity during sighs and airway occlusion compared with the preinjury levels.

Fig. 5.

Bilateral chronic diaphragm EMG activity for animals in the laminectomy (A; n = 9–12) and C4 unilateral contusion (B; n = 12) groups. Summary of diaphragm RMS EMG amplitude (RMS_peak) at preinjury (light gray) and 1 (dark gray) and 7 (black) days postinjury (DPI) normalized to the preinjury RMS EMG amplitude for sigh. In the repeated-measures analysis, there was an effect of group, behavior, and time. Animals in the laminectomy group displayed stable chronic diaphragm EMG values for all respiratory motor behaviors across the 8-day recording period. At 7 DPI, animals in the contusion group displayed significantly higher ipsilateral and contralateral RMS EMG amplitudes compared with the preinjury and 1 DPI values during occlusion and sighs (*post hoc Tukey-Kramer HSD at P < 0.05). HH, hypoxia-hypercapnia.

Fig. 6.

Estimated neural drive to phrenic motoneurons in the laminectomy (A; n = 9–12) and unilateral C4 contusion (B; n = 12) groups. Neural drive was defined as the RMS value at 75 ms after the onset of diaphragm activity (RMS₇₅) and was normalized to the preinjury RMS EMG amplitude during sigh. In the repeated-measures analysis, there was an effect of group, behavior, and time. Animals in the laminectomy group displayed stable chronic diaphragm RMS₇₅ EMG values for all respiratory motor behaviors across the 8-day recording period. Animals in the contusion group displayed RMS₇₅ EMG values during airway occlusion that were significantly higher at 7 DPI compared with preinjury and 1 DPI (*post hoc Tukey-Kramer HSD at P < 0.05).