Acute delivery of EphA4-Fc improves functional recovery after contusive spinal cord injury in rats

Abstract

Blocking the action of inhibitory molecules at sites of central nervous system injury has been proposed as a strategy to promote axonal regeneration and functional recovery. We have previously shown that genetic deletion or competitive antagonism of EphA4 receptor activity promotes axonal regeneration and functional recovery in a mouse model of lateral hemisection spinal cord injury. Here we have assessed the effect of blocking EphA4 activation using the competitive antagonist EphA4-Fc in a rat model of thoracic contusive spinal cord injury. Using a ledged tapered balance beam and open-field testing, we observed significant improvements in recovery of locomotor function after EphA4-Fc treatment. Consistent with functional improvement, using high-resolution ex vivo magnetic resonance imaging at 16.4T, we found that rats treated with EphA4-Fc had a significantly increased cross-sectional area of the dorsal funiculus caudal to the injury epicenter compared with controls. Our findings indicate that EphA4-Fc promotes functional recovery following contusive spinal cord injury and provides further support for the therapeutic benefit of treatment with the competitive antagonist in acute cases of spinal cord injury.

FIG. 1.

EphA4-Fc prevents ephrin-induced phosphorylation of EphA4. (A) Chinese hamster ovary (CHO) cells expressing EphA4 were blocked with EphA4-Fc prior to the addition of ephrin A4-Fc. Lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and bands specific for total EphA4 and phosphorylated EphA4 detected. (B) Quantification of band densities shows that only in the absence of EphA4-Fc could ephrin A4-Fc significantly increase the phosphorylation of EphA4 above basal levels (one-way ANOVA with Bonferroni's post-test). Blocking with EphA4-Fc prevented ephrin-induced phosphorylation of EphA4. ***p<0.001.

FIG. 2.

EphA4-Fc improves coordination and balance after contusive spinal cord injury (SCI). (A) At 120 days following injury, animals treated with 5 mg/kg or 20 mg/kg EphA4-Fc showed a significant improvement in their ability to cross the ledged, tapered beam before misstepping (one-way ANOVA with Dunnett's post-test). (B) Rats treated with 1 mg/kg or 20 mg/kg EphA4-Fc had significantly fewer falls than animals treated with the vehicle (one way ANOVA with Dunnett's post test; p=0.074 for animals treated with 5 mg/kg EphA4-Fc, unpaired t test). (C) When data from all EphA4-Fc treatment groups were pooled, the mean distance before a misstep for animals treated with EphA4-Fc was significantly greater than for animals treated with vehicle alone (unpaired t test). *p<0.05, **p<0.01; d.p.i.: days post-injury.

FIG. 3.

EphA4-Fc improves open-field locomotor performance after contusive spinal cord injury (SCI). (A) At 120 days following injury, rats treated with 1 mg/kg EphA4-Fc had significantly better Basso, Beattie and Bresnahan (BBB) scores than rats treated with the vehicle alone (one-way ANOVA with Dunnett's post-test). (B) When combined, all EphA4-Fc treated animals had significantly better open-field locomotor performance than did controls (unpaired t test). *p<0.05, **p<0.01; d.p.i.: days post-injury.

FIG. 4.

MRI analysis shows that the area of the dorsal funiculus is greater following EphA4-Fc treatment after spinal cord injury (SCI). Examples of high-resolution MRI mid-sagittal views of injured spinal cords from rats treated with (A) vehicle alone (n=7), (B) 1 mg/kg (n=7), (C) 5 mg/kg (n=6), and (D) 20 mg/kg EphA4-Fc (n=8). (E) Example of a coronal MRI slice (from [B], white line indicates location), with regions of interest (ROIs) shown in (F). DF: dorsal funiculus; GM: gray matter; VL: ventrolateral white matter; C: cyst; and D: damaged tissue. (G–I) The area of the dorsal funiculus in rats treated with (G) 1 mg/kg, (H) 5 mg/kg, and (I) 20 mg/kg EphA4-Fc was significantly greater caudal to the injury epicenter when compared with controls. The area of the dorsal funiculus for rats treated with the vehicle is shaded to the extent of its standard error of the mean. (J) A schematic of the increased area of the dorsal funiculus caudal to the injury epicenter (striped area) in EphA4-Fc-treated rats compared with rats treated with the vehicle alone. Scale bars for panels (A–D) as indicated in (A), and for (E) and (F) as indicated in (F). *p<0.05, **p<0.01, two-way ANOVA with Bonferroni's post-test.

FIG. 5.

Diffusion tensor imaging (DTI) analysis shows that the area of the dorsal funiculus is greater following EphA4-Fc treatment after spinal cord injury (SCI). (A–D) Example of a high-resolution DTI midsagittal images. Examples of the regions of interest (ROIs) from (A) are shown in (B–D) (rostral to caudal, B: −2.0 mm, C: 0.0 mm, D: +2.0 mm). (E) The cross-sectional area of the dorsal funiculus in the 5 mg/kg group was significantly greater 2 mm caudal to the injury epicenter, and in the 20 mg/kg EphA4-Fc treatment group, the area was significantly greater 3 mm rostral and 2–3.5 mm caudal to the lesion epicenter. Quantitative measures of diffusion (F, fractional anisotropy; G, apparent diffusion coefficient; H, axial diffusivity; and I, transverse diffusivity) reveal an increasing loss of white matter integrity closer to the lesion epicenter. The vehicle-treated group is shaded to the extent of its standard error of the mean. Scale bar for (A–D) as indicated in (A).+ p<0.05, *p<0.05, **p<0.01, ***p<0.001, two-way ANOVA with Bonferroni's post-test.

FIG. 6.

EphA4-Fc treatment improves open-field locomotor performance in the acute phase after injury. When tested in the open field, rats treated with 1 mg/kg or 5 mg/kg EphA4-Fc had significantly higher Basso, Beattie and Bresnahan (BBB) scores than did vehicle-treated animals (A) 1 day, (B) 3 days, and (C) 14 days after contusive injury (one-way ANOVA with Dunnett's post-test). *p<0.05, **p<0.01.

FIG. 7.

EphA4-Fc treatment did not affect the nociceptive response to pressure. Treated animals did not show any increase in mechanical hyperalgesia as tested using the (A) von Frey assay and (B) the Randall–Siletto assay. The animals treated with 5 mg/kg or 20 mg/kg EphA4-Fc were significantly less sensitive using the Randall–Siletto measure of broad pressure pain (one-way ANOVA with Dunnett's post-test). *p<0.05.