The Impact of Midcervical Contusion Injury on Diaphragm Muscle Function

Abstract

Midcervical contusion injuries disrupt descending ipsilateral excitatory bulbospinal projections to phrenic motoneurons, compromising ventilation. We hypothesized that a unilateral contusion injury at C3 versus C5 would differentially impact phrenic activity reflecting more prominent disruption of ipsilateral descending excitatory drive to more caudal segments of the phrenic motor pool with more cranial injuries. Phrenic motoneuron counts and evidence of diaphragm muscle denervation at individual neuromuscular junctions (NMJ) were evaluated at 14 days post-injury after unilateral contusion injury (100 kDynes). Whole body plethysmography and chronic diaphragm EMG were measured before the injury and at 3, 7, and 14 days post-injury. Contusion injuries at either level resulted in a similarly sized cavity. C3 contusion resulted in loss of 39 ± 13% of ipsilateral phrenic motoneurons compared with 13 ± 21% after C5 contusion (p = 0.003). Cervical contusion injuries resulted in diaphragm muscle denervation (C3 contusion: 17 ± 4%; C5 contusion: 7 ± 4%; p = 0.047). The pattern of denervation revealed segmental innervation of the diaphragm muscle, with greater denervation ventrally after C3 contusion and dorsally after C5 contusion. Overall, diaphragm root mean square electromyography activity did not change ipsilaterally after C3 or C5 contusion, but increased contralaterally (∼ 11%) after C3 contusion only on the first day post-injury (p = 0.026). Similarly, there were no significant changes in breathing parameters during eupnea or exposure to hypoxia (10% O2) - hypercapnia (5% CO2) at any time post-injury. Unilateral midcervical contusions minimally impair ventilatory behaviors despite phrenic motoneuron loss and diaphragm muscle denervation.

Keywords: level of injury; neuromuscular junction; phrenic motoneuron; spinal cord injury; ventilation.

FIG. 1.

Spinal cord histology after unilateral midcervical contusion injury. Representative 70 μm thick transverse spinal cord sections from an individual C3 and C5 contusion animal at 14 days post-injury. The level of maximum injury was the C3/C4 level for the C3 contusion and C5/C6 level for C5 contusion groups. In all cases, the pattern of cyst and scar formation compromised ventral and dorsal gray matter on the contused side only. A small notch on the left side was used to orient the sections (dorsal is on top). Bar, 1 mm.

FIG. 2.

Distribution of phrenic motoneurons after unilateral C3 or C5 contusion injury. Phrenic motoneuron distribution across spinal cord levels C3 to C6 was similar in all three groups on the contralateral (intact) side. Differences in motoneuron counts across sides were only evident in the C3 contusion group (two-way analysis of variance, p < 0.002). Error bars represent standard error.

FIG. 3.

Diaphragm neuromuscular junction (NMJ) denervation after unilateral C3 or C5 contusion injury. Representative maximum intensity projections from an innervated (A) and denervated (B) NMJ labeled pre-synaptically with synaptophysin (red) and post-synaptically with Alexa Fluor 488-conjugated α-bungarotoxin. Bar, 10 μm. (C) Intact diaphragm muscle showing midcostal and crural sections (crural at bottom). The midcostal diaphragm was divided into three zones in a ventral to dorsal direction in general agreement with predicted segmental innervation. (D) The percentage of NMJs showing denervation in the diaphragm overall as well as per zone in each group. NMJ denervation after C3 contusion was increased overall compared with sham/laminectomy (*, p = 0.047). In the C3 contusion group, NMJ denervation was increased in zone 1 compared with zone 3 (^†). In zone 1, NMJ denervation in the C3 contusion group was increased compared with the other two groups (^‡). No difference in the percent of NMJ denervation was evident between the C5 contusion and sham/laminectomy groups for any zone. No difference across groups was evident in other zones. Error bars represent standard error.

FIG. 4.

Morphometric assessment of NMJ denervation after unilateral C3 or C5 contusion injury. (A) Binarized images showing three-dimensional rendering of pre- and post-synaptic volumes for representative innervated (top) and denervated (bottom) NMJs in Figure 3, with varying depth represented by grayscale intensity (scale bar at right). Images represent the volume of apposition between pre- and post-synaptic structures, with depth shown by scale at right. Bar, 10 μm. (B) Cumulative probability plot for volume of apposition (as a percent of post-synaptic volume). Reduced pre- vs. post-synaptic apposition is evident for the C3 contusion group compared with other groups (left shifted distribution). (C) Volume of apposition across zones and groups. After C3 contusion, NMJs in zone 1 had lower volume of apposition than in zone 3 (*, p < 0.001). No other differences were evident across groups or zones. Error bars represent standard error.

FIG. 5.

Chronic diaphragm electromyographic (EMG) activity before and after unilateral C3 or C5 contusion injury. (A) Representative raw diaphragm EMG recordings and root mean square (RMS) EMG tracings from a C3 contusion animal monitored during sigh and eupnea at 0 and 1 days post-injury (DPI). (B) Diaphragm RMS EMG amplitude at 0, 1, 3, 7, and 14 DPI normalized to RMS EMG amplitude during sigh at 0 DPI. At 1 DPI, C3 contusion resulted in increased contralateral RMS EMG amplitude compared with laminectomy or C5 contusion groups (*, p = 0.026). No changes in RMS EMG amplitude were observed over time on the ipsilateral side in any group.

FIG. 6.

Tidal volume (V_T) measurements using whole body plethysmography at 0, 3, 7, and 14 days post-injury. No changes in V_T were observed over time after unilateral C3 or C5 contusion injury compared with the laminectomy group. All three groups showed a two-fold increase from eupnea to hypoxia-hypercapnia on all days evaluated. Data shown as mean ± standard error.

FIG. 7.

Locomotion analysis after unilateral C3 or C5 contusion injury, as measured by the 21-point Basso, Beattie and Bresnahan (BBB) scale. Animals were measured daily from 0 to 14 days post-injury (DPI). Both C3 and C5 contusion groups had lower scores compared with the laminectomy group from 1 to 9 DPI (p < 0.001). There was no difference in BBB scores between the two contused groups at any time post-injury. Data shown as mean ± standard error.