Article 2: Inhibition of Neurofilament Synthesis by Morpholino Antisense Oligonucleotides Inhibits Spinal Cord Axon Regeneration

Objective: To determine the role of neurofilament in central nervous system (CNS) axon regeneration. Design: Not provided. Setting: Not provided. Animals: Lampreys. Intervention: Fluorescently labeled antisense morpholinos to the lamprey neurofilament NF180 were delivered to brainstem neurons by retrograde transport from a fresh spinal cord transection. After 2 or 4 weeks (times of maximum axon retraction and regeneration back to the lesion, respectively), the CNS was dissected and axon tip locations recorded. Then brains were either prepared for neurofilament immunohistochemistry or homogenized for Western blotting. Main Outcome Measures: Animals were compared with those receiving a control morpholino. To determine the effect of morpholinos on regeneration past the lesion, 6 animals (3 anti-NF180, 3 control morpholino-treated) were allowed to recover 9 weeks, at which time neurons with regenerated axons were retrogradely labeled by a second dye applied to a second transection 5mm caudal to the first. Results: Axon retraction ± standard error of the mean at 2 weeks post-transection was 1.59±0.12mm in controls and 1.34±0.10mm with anti-NF180 morpholinos (t test, P<0.1). At 4 weeks, the distances of axon tips from the transection site were .32±.06mm and .57±.06mm, respectively (P<.01), translating into regeneration rates of 91.1 and 54.5μm/d for control and antisense morpholino groups, respectively. At both recovery times, the antisense treated brains showed reduced NF180 content. After 9 weeks in recovery, the anti-NF180 treated animals had 25% fewer regenerated neurons than the controls (t test, P<.05). Conclusions: The findings are consistent with a role for neurofilaments in the mechanism of axon regeneration in the CNS.