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, 29 (18), 2748-60

Degeneration of Phrenic Motor Neurons Induces Long-Term Diaphragm Deficits Following Mid-Cervical Spinal Contusion in Mice

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Degeneration of Phrenic Motor Neurons Induces Long-Term Diaphragm Deficits Following Mid-Cervical Spinal Contusion in Mice

Charles Nicaise et al. J Neurotrauma.

Abstract

A primary cause of morbidity and mortality following cervical spinal cord injury (SCI) is respiratory compromise, regardless of the level of trauma. In particular, SCI at mid-cervical regions targets degeneration of both descending bulbospinal respiratory axons and cell bodies of phrenic motor neurons, resulting in deficits in the function of the diaphragm, the primary muscle of inspiration. Contusion-type trauma to the cervical spinal cord is one of the most common forms of human SCI; however, few studies have evaluated mid-cervical contusion in animal models or characterized consequent histopathological and functional effects of degeneration of phrenic motor neuron-diaphragm circuitry. We have generated a mouse model of cervical contusion SCI that unilaterally targets both C4 and C5 levels, the location of the phrenic motor neuron pool, and have examined histological and functional outcomes for up to 6 weeks post-injury. We report that phrenic motor neuron loss in cervical spinal cord, phrenic nerve axonal degeneration, and denervation at diaphragm neuromuscular junctions (NMJ) resulted in compromised ipsilateral diaphragm function, as demonstrated by persistent reduction in diaphragm compound muscle action potential amplitudes following phrenic nerve stimulation and abnormalities in spontaneous diaphragm electromyography (EMG) recordings. This injury paradigm is reproducible, does not require ventilatory assistance, and provides proof-of-principle that generation of unilateral cervical contusion is a feasible strategy for modeling diaphragmatic/respiratory deficits in mice. This study and its accompanying analyses pave the way for using transgenic mouse technology to explore the function of specific genes in the pathophysiology of phrenic motor neuron degeneration and respiratory dysfunction following cervical SCI.

Figures

FIG. 1.
FIG. 1.
Diaphragm electrophysiology following double C4+C5 unilateral cervical contusion spinal cord injury (SCI). Compared with laminectomy control animals (A), C4+C5 injury mice (B) had reduced phrenic nerve compound muscle action potential (CMAP) amplitudes, a functional electrophysiological assay of phrenic nerve innervation of the diaphragm. Significant decreases in peak CMAP amplitudes were found in injured mice at 2 and 6 weeks post-injury (C). Spontaneous diaphragm electromyography (EMG) recordings showed similar motor unit potential activation amplitudes between laminectomy (D) and injured (E and F) mice. Representative traces are from C4+C5 mice at 2 weeks post-injury (E) or 6 weeks post-injury (F). The time scale and amplitude for the traces are indicated. A slight decrease in inspiratory burst frequency was observed in injured animals, becoming significant at 6 weeks post-injury (G). Quantitative analysis of EMG signals also showed an increase in the duration of each discharge in injured animals at 6 weeks post-injury (H). Results are expressed as means±SEM. *C4+C5 injury versus laminectomy (p<0.05); **C4+C5 injury versus laminectomy (p<0 .01). n=8–10 animals per group.
FIG. 2.
FIG. 2.
Histological analyses of spinal cord following double C4+C5 unilateral cervical contusion spinal cord injury (SCI). Representative images illustrate the rostral boundary of the lesion at the C3 level (C3: A), the impact at C4 (C4: B), the lesion epicenter at C5 (C5: C), and the caudal edge at the C6 level (C6: D) at 2 weeks post-injury. The typical lesion in mice affected most of the gray matter at the epicenter. The lesion also disrupted several white matter regions, extending along the dorsolateral, lateral, and ventrolateral funiculi at C4 and reaching the ventral funiculus at C5. Evidence of white matter degeneration was observed in the lateral funiculus at C3 and C6 levels. The uninjured group did not show any pathological abnormalities following the laminectomy procedure (C4: E). Illustrations are representative of 5–7 independent mice in each group (scale bar: 0.5 mm). Higher magnification of the lesion close to the epicenter is provided, showing the central canal (cc) and the ipsilateral ventral horn (VH) gray matter (scale bar: 100 μm) (F). Both gray and white matter regions were disrupted, hypercellularized and filled with dense fibrous tissue. Cresyl violet-stained motor neurons are seen in uninjured animals at all cervical regions (inset in E), whereas they are completely lost at the lesion epicenter (F). Quantitative lesion analyses did not show any significant difference in lesion extension or total lesion volume (G) between 2 and 6 weeks post-injury. Overall, the C4+C5 injury paradigm produced a lesion spreading over 3.5 mm in the rostral-caudal axis. Quantitative analyses of myelin loss did not show any extension of white matter damages beyond the lesion or any difference between 2 and 6 weeks post-injury (H). n=5–7 animals per group.
FIG. 3.
FIG. 3.
Quantification of total cervical motor neuron and phrenic motor neuron loss. Large motor neurons were identified on cresyl violet stained sections and were manually counted in the ipsilateral or contralateral ventral horns (VH) following C4+C5 injury (A and B). Following injury, a significant loss of motor neurons over a length of 4 mm was observed surrounding the lesion epicenter in the ipsilateral VH (A). Contralateral hemi-spinal cord remained unaffected, except at two locations opposite the lesion epicenter, where the mean number of motor neurons was slightly reduced (B). There were no differences in motor neuron counts between 2 weeks (gray square) and 6 weeks (black dot) post-injury. The number of motor neurons in the cervical spinal cord from uninjured laminectomy-only mice is denoted by a black triangle. The phrenic motor neuron pool was visualized specifically in the cervical spinal cord using fluorescently labeled CTβ retrograde tracer injected in the intrapleural space of the ipsilateral hemi-diaphragm (C-H). Representative illustrations show transverse (C) and longitudinal (D) sections of the cervical spinal cord, revealing cholera toxin β (CTβ)-labeled motor neurons (white arrow) in the VH. Inset in D: higher magnification image of CTβ+ phrenic motor neurons forming a compact and linear column. Surrounding the lesion epicenter, an obvious difference in the number of CTβ -labeled cells was observed between laminectomy (E) and injured (F) mice at 6 weeks post-injury. Bars represent 500 μm in C and D and 100 μm in E and F. Quantification of CTβ-labeled cells at multiple distances from the lesion epicenter showed significant loss of phrenic motor neurons at multiple locations within the ipsilateral lesioned hemi-spinal cord at 6 weeks post-injury (G). Quantification of CTβ-labeled cells shows a total loss of 56% of the phrenic motor neuron pool in the ipsilateral lesioned hemi-spinal cord at 6 weeks post-injury (H). Results are expressed as means±SEM. $C4+C5 injury versus laminectomy (p<0.05); &C4+C5 injury versus laminectomy (p<0.01); #C4+C5 injury versus laminectomy (p<0.001); **C4+C5 injury versus laminectomy at 6 weeks (p<0.01). n=5–7 animals per group.
FIG. 4.
FIG. 4.
Histological analysis of phrenic nerve. Phrenic nerves from C4+C5 injured (B) animals exhibited significant pathological changes such as degenerating fibers, proliferating Schwann cells, and endoneurial fibrosis, whereas abundant and compact myelinated fibers were found in the laminectomy control mice (A). The illustration in B is representative of 2 weeks post-injury, and a similar pattern of axonopathy was also observed at 6 weeks. Reduced density of myelinated axons was observed in ipsilateral phrenic nerves from injured mice at both time points compared with the laminectomy group (C). The average axonal area did not change significantly across conditions (D). Ultrastructural analysis of phrenic nerve sections from injured mice (F) shows degenerating axons surrounded by disrupted myelin sheath, compared with the normal morphology of axons in the laminectomy sham group (E). Bars represent 10 μm in A and B and 1 μm in E and F. Results are expressed as means±SEM. *C4+C5 injury versus laminectomy (p<0.05); n=5–7 animals per group.
FIG. 5.
FIG. 5.
Diaphragm histology and neuromuscular junction (NMJ) analysis. Hematoxylin and eosin staining did not show any observable pathological changes in ipsilateral hemidiaphragm muscle following C4+C5 injury (B) compared with laminectomy-only mice (A). Bars represent 200 μm in A and B. For quantitative analysis, three sections along the medial-lateral axis of the ipsilateral hemidiaphragm were analyzed per animal. On each section, the average of five thickness points equally distributed along the ventro-dorsal axis was measured in a blind manner (C). Global muscle thickness (D) and myofiber size (E) did not show any changes at 2 or 6 weeks post-injury. Diaphragm NMJs were assessed across three different regions of the ipsilateral hemidiaphragm (F) via labeling with rhodamine-alpha-bungarotoxin (red), SMI-312R (green) and SV2-s (green). Nearly all NMJs were completely intact in uninjured laminectomy mice (H and I), characterized by: complete overlap of the pre-synaptic axon and pre-synaptic vesicles with post-synaptic acetylcholine receptors, no signs of multiple innervations, and absence of pre-synaptic axon thinning. Double C4+C5 injury mice had strikingly fewer intact junctions at both time points (G) and displayed obvious areas of denervation in the hemidiaphragm (J). To specifically examine the types of morphological changes occurring in the hemidiaphragm muscle, NMJ changes were broken down into a number of phenotypic categories: intact (I), partially denervated (K), fully denervated (L), and signs of reinnervation supported by the presence of multiply innervated NMJs (M) and by thin preterminal axons (N). Panels J and L provide an example of a fully denervated NMJ (#1) and a partially denervated NMJ (#2). Both types of denervated junctions were only found in C4+C5 injured mice at both 2 and 6 weeks post-injury, mostly in the central and dorsal regions of the hemidiaphragm. Very rare instances of junctional pathologies were found in the laminectomy sham group. Details about the NMJ phenotypes are found in Table 3. Bars represent 20 μm in H-N. Results are expressed as means±SEM. **C4+C5 injury at 2 weeks versus laminectomy (p<0.01); ***C4+C5 injury at 2 weeks versus laminectomy (p<0.001); #C4+C5 injury at 6 weeks versus laminectomy (p<0.05); ###C4+C5 injury @ 6 weeks versus laminectomy (p<0.001). n=3 animals in all conditions. Color image is available online at www.liebertonline.com/neu.

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