Axonal regeneration by chronically-injured supraspinal neurons can be enhanced by neurotrophic factor treatment at the site of injury, although the number of regenerating neurons decreases as the interval between spinal cord injury and treatment increases. This study investigated whether this decline in regenerative response could be due to continued loss of neurons during the post-injury period. Adult rats received a cervical hemisection lesion and axotomized neurons were labeled by retrograde transport of True Blue from the lesion site. Animals were killed one, four or eight weeks after injury and surviving neurons (True Blue-labeled) were counted in the red nucleus and lateral vestibular nucleus. The neuron number in the lateral vestibular nucleus was stable for eight weeks after spinal cord injury, while survival in the red nucleus decreased by 25% between four and eight weeks. To test how neurons respond to a second injury with or without trophic factor treatment, at four, eight, 14 or 22 weeks after injury the lesion cavity was enlarged by 0.5 mm in a rostral direction. Gel foam saturated with ciliary neurotrophic factor, brain-derived neurotrophic factor or basic fibroblast growth factor was placed into the cavity. Animals were killed four weeks later. Re-injury of the spinal cord caused a significant decrease in neuron survival in both the red nucleus and lateral vestibular nucleus, the effects of which were lessened by treatment with ciliary neurotrophic factor or brain-derived neurotrophic factor for the red nucleus and with ciliary neurotrophic factor for the lateral vestibular nucleus, when re-injured at four or eight weeks. Basic fibroblast growth factor did not affect neuron survival at any time post-injury. Ciliary neurotrophic factor was not effective with longer delays (14 or 22 weeks) between the initial injury and re-injury. These results indicate a delayed pattern of secondary neuronal cell loss after spinal cord injury that is exaggerated by re-injury, but which can be ameliorated by treatment with neurotrophic factors.