Rho signaling pathway targeted to promote spinal cord repair

Abstract

The Rho signaling pathway regulates the cytoskeleton and motility and plays an important role in neuronal growth inhibition. Here we demonstrate that inactivation of Rho or its downstream target Rho-associated kinase (ROK) stimulated neurite growth in primary cells of cortical neurons plated on myelin or chondroitin sulfate proteoglycan substrates. Furthermore, treatment either with C3 transferase (C3) to inactivate Rho or with Y27632 to inhibit ROK was sufficient to stimulate axon regeneration and recovery of hindlimb function after spinal cord injury (SCI) in adult mice. Injured mice were treated with a single injection of Rho or Rho-associated kinase inhibitors delivered in a protein adhesive at the lesion site. Treated animals showed long-distance regeneration of anterogradely labeled corticospinal axons and increased levels of GAP-43 mRNA in the motor cortex. Behaviorally, inactivation of Rho pathway induced rapid recovery of locomotion and progressive recuperation of forelimb-hindlimb coordination. These findings provide evidence that the Rho signaling pathway is a potential target for therapeutic interventions after spinal cord injury.

Fig. 1.

Effect of Rho antagonist C3 or Rho-associated kinase inhibitor Y27632 on neurite outgrowth of primary cortical neurons plated on inhibitory substrates. A, Neurite outgrowth was analyzed quantitatively by measuring the longest neurite per cell 24 hr after plating on poly-l-lysine, myelin, CSPG, or mixed substrates and treatment with buffer (white), 25 μg/ml C3 (gray), or 31 μm Y27632 (black). Differences between treated and untreated cells were significant on all test substrates (t test; p < 0.05).B–D, Representative micrographs of cortical neurons plated on mixed inhibitory substrates either untreated (B) or treated with C3 (C) or Y27632 (D). Scale bar, 25 μm.

Fig. 2.

Axon regeneration after SCI and treatment with C3. Shown are dark-field micrographs of spinal cord sections; rostral is to the left. A, Anterogradely labeled CST fibers in a C3-treated spinal cord 3 weeks after injury. The lesion site is between the dotted lines. B, Same section asA 12 mm caudal to the lesion site. C, Higher magnification of boxed region in Ato show regenerative sprouting into lesion site and into the dorsal white matter. D, A different section from the same animal as A, showing regenerating fibers 10 mm from the lesion site. E, Anterogradely labeled CST fibers 3 months after SCI sprout into the dorsal white matter and cross the lesion site. The lesion appears as a vertical line; accumulated blood contributes to the bright appearance of the lesion.F, Same section as E taken 8 mm from the lesion site. G, Fibrin-treated control showing some sprouting of lesioned fibers, but no long-distance regeneration.H, Anterogradely labeled fibers retract from the lesion site in an untreated animal. I, Neutral red staining showing glial scar 1 month after lesion. Arrows indicate axon sprouting. Scale bars: A, B,E, G, H, 500 μm;I, 250 μm; C,D, 100 μm; F, 25 μm.

Fig. 3.

Quantification of regeneration length. Longest regeneration distances after SCI alone or treatment with vehicle, Rho antagonist C3, or Rho kinase inhibitor Y27632. Each point represents one animal. The circles represent animals examined 3 weeks to 1 month after SCI; the triangles represent animals examined at 3 months. Lines indicate averages for each group. Statistical significances were evaluated with the unpaired t test: C3 + collagen versus collagen,p < 0.05; C3 + fibrin versus fibrin,p < 0.001; Y27632 + fibrin versus fibrin,p < 0.05; C3 + collagen versus C3 + fibrin,p < 0.05; C3 + fibrin versus Y27632 + fibrin,p < 0.01.

Fig. 4.

Expression of GAP-43 mRNA in motor cortex.A, Photomicrograph of a cresyl violet-stained coronal section of mouse brain. Box outlines motor cortex area depicted in B–D. B, Fluorescent micrograph of same section as in A showing neurons retrogradely labeled with Fluorogold applied at the site of a dorsal hemisection at T7. C, D, Dark-field photomicrographs showing sections of motor cortex from an untreated mouse (C) or a C3/fibrin-treated mouse (D) after in situhybridization for GAP-43. E, Quantitation showing significantly increased grain density in motor cortex after treatment with C3. Differences between C3 and background (Bkgd) were significant (t test; p < 0.05); differences between PBS and background were not significant. Scale bar: A, 1.8 mm; B, 1.2 mm;C, D, 250 μm.

Fig. 5.

Analysis of functional recovery. A, Modified BBB scores of C3-treated (black circles;n = 11), Y27632-treated (black triangles; n = 5), fibrin-treated (gray circles; n = 11), and untreated (open circles; n = 10) mice to evaluate recovery of locomotion during the month after dorsal over-hemisection. At 24 hr, 1 week, 2 weeks, and 1 month, differences between groups of animals were evaluated by the Mann–WhitneyU test. p values were similar at all four time points: C3-treated versus fibrin-treated, p < 0.001; Y27632-treated versus fibrin-treated, p < 0.05; C3-treated versus Y27632-treated, NS; fibrin-treated versus untreated, NS. B, Photograph of a spinal cord-injured mouse 24 hr after injury; HL cannot support body weight.C, Photograph of a C3/fibrin-treated mouse 24 hr after injury; body weight is supported by HL. D,E, Selected videoframes of representative untreated and C3/fibrin-treated mice, respectively, to show differences in recovery of HL–FL coordination 30 d after lesion. Although C3-treated mice alternate front paw and foot placements properly, untreated mice do not show one-to-one correspondence between HL and FL stepping.Numbers refer to elapsed time in tenths of seconds.F, G, For the mice depicted inD and E, respectively, HL–FL coordination is represented graphically. The position of the right hindpaw (solid line) and the right front paw (dotted line) on (I) or off (O) the ground was noted for each 1 of 38 sequential videoframes. The untreated mouse in D moves its forelimb twice before each HL step. NS, Not significant.