Regenerative growth of corticospinal tract axons via the ventral column after spinal cord injury in mice.

Studies that have assessed regeneration of corticospinal tract (CST) axons in mice after genetic modifications or other treatments have tacitly assumed that there is little if any regeneration of CST axons in normal mice in the absence of some intervention. Here, we document a previously unrecognized capability for regenerative growth of CST axons in normal mice that involves growth past the lesion via the ventral column. Mice received dorsal hemisection injuries at thoracic level 6-7, which completely transect descending CST axons in the dorsal and dorsolateral column. Corticospinal projections were traced by injecting biotinylated dextran amine (BDA) into the sensorimotor cortex of one hemisphere either at the time of the injury or 4 weeks after injury, and mice were killed at 20-23 or 46 d after injury. At 20-23 d after injury, BDA-labeled CST axons did not extend past the lesion except in one animal. By 46 d after injury, however, a novel population of BDA-labeled CST axons could be seen extending from the gray matter rostral to the injury into the ventral column, past the lesion, and then back into the gray matter caudal to the injury in which they formed elaborate terminal arbors. The number of axons with this highly unusual trajectory was small ( approximately 1% of the total number of labeled CST axons rostral to the injury). The BDA-labeled axons in the ventral column were on the same side as the main tract and thus are not spared ventral CST axons (which would be contralateral to the main tract). These results indicate that normal mice have a capacity for CST regeneration that has not been appreciated previously, which has important implications in studying the effect of genetic or pharmacological manipulations on CST regeneration in mice.

Forelimb locomotor assessment scale (FLAS): novel assessment of forelimb dysfunction after cervical spinal cord injury.

We describe here a novel forelimb locomotor assessment scale (FLAS) that assesses forelimb use during locomotion in rats injured at the cervical level. A quantitative scale was developed that measures movements of shoulder, elbow, and wrist joints, forepaw position and digit placement, forelimb-hindlimb coordination, compensatory behaviors adopted while walking, and balance. Female Sprague-Dawley rats received graded cervical contusions ranging from 200 to 230 ("mild," n=11) and 250-290 kdyn ("moderate," n=13) between C5 and C8. Rats were videotaped post-injury as they walked along an alley to determine deficits and recovery of forelimb function. Recovery of shoulder and elbow joint movement occurred rapidly (within 1-7 days post-injury), whereas recovery of wrist joint movement was slower and more variable. Most rats in all groups displayed persistent deficits in forepaw and digit movement, but developed compensatory behaviors to allow functional forward locomotion within 1-2 weeks post-injury. Recovery of forelimb function as measured by the FLAS reached a plateau by 3 weeks post-injury in all groups. Rats with mild contusions displayed greater locomotor recovery than rats with moderate contusions, but exhibited persistent deficits compared to sham controls. Reliability was tested by having seven raters (three internal, four external) from different laboratories, independently and blindly score videos of all rats. The multivariate correlation between all raters, all animals, and all time points ranged from r(2)=0.88-0.96 (p<0.0001), indicating a high inter-rater reliability. Thus, the FLAS is a simple, inexpensive, sensitive, and reliable measure of forelimb function during locomotion following cervical SCI.

A re-assessment of the effects of a Nogo-66 receptor antagonist on regenerative growth of axons and locomotor recovery after spinal cord injury in mice.

This study was undertaken as part of the NIH "Facilities of Research-Spinal Cord Injury" project to support independent replication of published studies. Here, we repeated a study reporting that treatment with the NgR antagonist peptide NEP1-40 results in enhanced growth of corticospinal and serotonergic axons and enhanced locomotor recovery after thoracic spinal cord injury. Mice received dorsal hemisection injuries at T8 and then received either NEP1-40, Vehicle, or a Control Peptide beginning 4-5 h (early treatment) or 7 days (delayed treatment) post-injury. CST axons were traced by injecting BDA into the sensorimotor cortex. Serotonergic axons were assessed by immunocytochemistry. Hindlimb motor function was assessed using the BBB and BMS scales, kinematic and footprint analyses, and a grid climbing task. There were no significant differences between groups in the density of CST axon arbors in the gray matter rostral to the injury or in the density of serotonergic axons caudal to the injury. Tract tracing revealed that a small number of CST axons extended past the lesion in the ventral column in some mice in all treatment groups. The proportion of mice with such axons was higher in the NEP1-40 groups that received early treatment. In one experiment, mice treated with either NEP1-40 or a Control Peptide (reverse sequence) had higher BBB and BMS scores than Vehicle-treated controls at the early post-injury testing intervals, but scores converged at later intervals. There were no statistically significant differences between groups on other functional outcome measures. In a second experiment comparing NEP-treated and Vehicle controls, there were no statistically significant differences on any of the functional outcome measures. Together, our results suggest that treatment with NEP1-40 created a situation that was slightly more conducive to axon regeneration or sprouting. Enhanced functional recovery was not seen consistently with the different functional assessments, however.

A re-assessment of the consequences of delayed transplantation of olfactory lamina propria following complete spinal cord transection in rats.

This study is part of the NIH "Facilities of Research-Spinal Cord Injury" contract to support independent replication of published studies. We repeated a study reporting that delayed transplantation of olfactory lamina propria (OLP) into the site of a complete spinal cord transection led to significant improvement in hindlimb motor function and induced axon regeneration. Adult female rats received complete spinal cord transections at T10. Thirty days post-injury, pieces of OLP, which contains olfactory ensheathing cells (OECs), or respiratory lamina propria (RLP), which should not contain OECs, were placed into the transection site. Hindlimb motor function was tested using the BBB scale from day 1 post-injury through 10 weeks following transplantation. To assess axonal regeneration across the transection site, Fluorogold was injected into the distal segment, and the distribution of 5HT-containing axons was assessed using immunostaining. BBB analyses revealed no significant recovery after OLP transplantation and no significant differences between OLP vs. RLP transplant groups. Fluorogold injections into caudal segments did not lead to retrograde labeling in any animals. Immunostaining for 5HT revealed that a few 5HT-labeled axons extended into both RLP and OLP transplants and a few 5HT-labeled axons were present in sections caudal to the injury in 2 animals that received OLP transplants and 1 animal that received RLP transplants. Our results indicate that, although OLP transplants may stimulate regeneration under some circumstances, the effect is not so robust as to reliably overcome the hostile setting created by a complete transection paradigm.