FIG. 1.

Early time course of histopathology following unilateral C4 cervical contusion. Eriochrome R/Nissl staining shows the lesion epicenter at 1 day post-injury (DPI) (A). Evidence of hemorrhage and edema is observed in ipsilesioned hemicord (arrowheads in A). Loss of large Nissl⁺ motor neurons in ipsilateral ventral horn was observed in addition to white matter disruption in dorsolateral, lateral and ventrolateral funiculi (scale bar: 1mm). The lesion size expanded over time, particularly at the epicenter (B). There was a significant increase in total lesion volume at 4 DPI, likely because of spinal cord swelling (C). Three-dimensional (3-D) reconstruction of the cervical spinal cord shows that the injury damaged both gray and white matter (D). At 1 DPI, there was significant extension of the lesion in rostral and caudal directions from the epicenter. At 14 DPI, there was still significant rostral-caudal extension; however, the lesion became more concentrated to the contusion epicenter (blue: entire spinal cord; red: gray matter; green: lesion). After C4 injury, a significant loss of motor neurons over a rostral-caudal length of ∼ 4 mm was observed surrounding the lesion epicenter (E). There were no differences among all time points from 1 to 14 DPI. The phrenic motor neuron (PhMN) pool was specifically labeled with cholera toxin β (CTβ)-Alexa555 via retrograde tracing from the diaphragm. CTβ⁺ PhMNs were identified in the area of the phrenic nucleus: mid-C3 to the rostral part of C5 (F). Representative illustrations show a longitudinal section of the phrenic nucleus following C4 injury (asterisk in F) and a transverse section from an uninjured control animal (Inset in F). Bars represent 500 μm in F. Numbers of CTβ⁺ PhMNs were quantified in transverse sections (inset in F). In the ipsilesioned hemicord, there was a significant loss of CTβ⁺ PhMNs at multiple distances from the lesion epicenter compared with injured controls (G) at 600 μm rostral, at epicenter, at 600 μm caudal, and up to 1200 μm caudal. Compared with 1 DPI, additional PhMN loss occurred rostrally at 1200 μm at 4 DPI, 8 DPI, and 14 DPI. As early as 4 DPI, phrenic nerves from C4 injured animals exhibited histopathological changes such as degenerating fibers (arrowheads in H). Bar represents 50 μm in H. Axonal density (i.e., number of myelinated fibers per unit area) was significantly decreased at 14 DPI compared with the uninjured group (I). Results are expressed as means±SEM. Statistical significance was assessed by analysis of variance (one-way ANOVA) and multiple comparisons post-hoc test (Bonferroni's method). *DPI versus uninjured (p<0.05); **DPI versus uninjured (p<0.01); ***DPI versus uninjured (p<0.001). n=5 animals for uninjured group and for injured animals at 1, 8, and 14 DPI; n=9 animals for injured animals at 4 DPI. Color image is available online at www.liebertpub.com/neu