Complement plays an important role in spinal cord injury and represents a therapeutic target for improving recovery following trauma

Abstract

Initiation of an inflammatory cascade following traumatic spinal cord injury (SCI) is thought to cause secondary injury and to adversely impact functional recovery, although the mechanisms involved are not well defined. We report on the dynamics of complement activation and deposition in the mouse spinal cord following traumatic injury, the role of complement in the development of SCI, and the characterization of a novel targeted complement inhibitor. Following traumatic injury, mice deficient in C3 had a significantly improved locomotor score when compared with wild-type controls, and analysis of their spinal cords revealed significantly more tissue sparing, with significantly less necrosis, demyelination, and neutrophil infiltration. Wild-type mice were also treated with CR2-Crry, a novel inhibitor of complement activation that targets to sites of C3 deposition. A single intravenous injection of CR2-Crry 1 hour after traumatic injury improved functional outcome and pathology to an extent similar to that seen in C3-deficient animals. CR2-Crry specifically targeted to the injured spinal cord in a distribution pattern corresponding to that seen for deposited C3. As immunosuppression is undesirable in patients following SCI, targeted CR2-Crry may provide appropriate bioavailability to treat SCI at a dose that does not significantly affect systemic levels of serum complement activity.

Figure 1

Complement activation as shown by C3 deposition at the site of spinal cord injury. 1 hour (A), 24 hours (B), and 72 hours (C). C3 deposition is marked by green fluorescent staining of the polyclonal anti-mouse C3 antibody in spinal cords harvested at different time points after injury (n = 3). In contrast laminectomy sham controls lack immunoreactivity for C3 (D).

Figure 2

Combined BBB locomotor scores post-SCI within sham, vehicle control, C3-deficient, and CR2-Crry groups. Note significant improvement in BBB score at day 3, 7, and 21 in both the C3-deficient and CR2-Crry groups when compared to vehicle controls (P = 0.001) (n = 12). The values are expressed as mean ± SE.

Figure 3

Tissue sparing as assessed by analyzing the cross-sectional area of spinal cords removed from vehicle controls, C3-deficient, and CR2-Crry-treated animals at 24 hours (upper panel), 72 hours (middle panel), and 7 days after injury (lower panel). Measurements were made from histological sections taken at 100-μm increments extending 2 mm either side of the injury site. No significant difference in tissue sparing was evident at 24 and 72 hours. Significant tissue sparing was noted in CR2-Crry and C3-deficient animals compared to vehicle controls at day 7 (P = 0.002). Mean ± SD, n = 4.

Figure 4

H&E-stained sections of spinal cord centered on the injury site at days 7 and 21 after injury. A–B: vehicle control. C–D: C3-deficient animals. E–F: CR2-Crry-treated animals. Original magnification, ×100.

Figure 5

Luxol fast blue stained section of spinal cord centered on the injury site at days 7 and 21 after injury. A–B: vehicle control. C–D: C3-deficient animals. E–F: CR2-Crry-treated animals. Original magnification, ×100.

Figure 6

Neutrophil infiltration as assessed by immunofluorescent staining with anti-mouse GR1-fluorescein labeled antibody. The total number of neutrophils per section was counted. The number of neutrophils is significantly lower in C3-deficient and CR2-Crry-treated animals across all time points when compared with vehicle control (* P = 0.001). Mean ± SD, n = 5 per group.

Figure 7

A: Biodistribution of CR2-Crry following spinal cord injury. ¹²⁵I CR2-Crry was injected into normal C57Bl/6 mice (no injury) and C57Bl/6 animals 1 hour following spinal cord injury. Tissue distribution was assessed 12 hours after initial injury in major organs and tissues of the central nervous system. B: Immunolocalization of CR2-Crry binding with an anti-CR2 Ab. Positive immunoflourescence is noted in the white matter beneath the injury site with a pattern similar to that observed in C3 stained sections (arrows). C: CR2 immunoreactivity seen in neuronal cells within the dorsal horn of the gray matter (arrows). Images are taken under oil immersion using ×63 objective and are representative of n = 4.