Axonal regeneration and functional recovery in the chick following embryonic spinal cord injury

The embryonic central nervous system (CNS) is more plastic than adult CNS, and the early embryonic brain and spinal cord has been suggested to recover more readily from a severe injury. The following studies were designed to determine the stages of chicken (Gallus domesticus) embryonic development during which descending brainstem-spinal tracts maintain their capacity for anatomical and functional repair after complete thoracic spinal cord transection. Brainstem-spinal neurons begin to project their axons through the spinal cord at approximately embryonic day (E)4, and these projections are essentially complete to the lumbar level by E12. Disruption of these brainstem-spinal pathways was produced by complete thoracic spinal cord transections at different developmental ages from E3 through E14. Control sham operations were conducted in parallel. The post-operative embryonic recovery period varied from 5 to 8 days. Following recovery, the extent of anatomical repair was assessed by injecting a fluorescent dye into the lumbar spinal cord caudal to the transection site. Brainstem tissue sections were subsequently examined for the presence of retrogradely labelled brainstem-spinal neurons. Anatomical results indicated similar distributions of retrogradely labelled neurons within the brainstem of both sham transected controls and embryos transected prior to E13 (Hasan et al., 1991, 1992). The neuroanatomical recovery shown by chick embryos can be attributed either to axonal regeneration of previously severed axons or to the subsequent development of new axonal projections from later developing neurons. In order to address this issue, the embryonic lumbar spinal cord was injected before and after thoracic transection with two different retrograde tract tracing fluorescent dyes. Co-localization of both labels within the same brainstem-spinal neuron would be indicative of regeneration of previously axotomized projections rather than the subsequent development of new axonal projections. Findings indicated that there were double-labelled brainstem—spinal neurons after a transection prior to E13 and the number of double-labelled brainstem-spinal neurons decreased after an E13-E15 transection. In addition, at each subsequent stage of development from E1O-E12, a higher ratio of double-labelled brainstem-spinal neurons (indicating regeneration of previously severed axons) to the number of cell bodies labelled with the second fluorescent tracer alone (indicating possible subsequent development) was observed. This would suggest that during successive stages of development, regeneration of previously axotomized fibers increasingly contributes to the observed anatomical and functional recovery after thoracic cord transections prior to E13 (Hasan et al., 1992). Functional recovery was assessed by focal electrical stimulation of identified brainstem locomotor regions in transected or sham-transected E18-E20 embryos. Leg muscle electromyographic (EMG) recordings were used to monitor brainstem stimulated locomotor activity (Valenzuela et al., 1990; Hasan et al., 1991, 1992). Functional repair was evident among E18-E20 embryos that had had their spinal cords transected prior to E13 and this brainstem evoked locomotion was indistinguishable from brainstem-evoked locomotion in control (sham-transected or untransected) embryos. In addition, voluntary open-field locomotion and brainstem evoked locomotion in hatchling chicks transected prior to E13 was indistinguishable from that observed in control hatchlings, indicating that complete functional recovery had occurred. Embryos and hatchling chicks transected on or after E13 showed reduced functional repair abilities (Hasan et al., 1991, 1992.Medicine, Faculty ofGraduat