Characterization of a period of vulnerability to the toxic effects of colchicine during regeneration of the goldfish optic nerve.

Abstract

The effects of intraocular (i.o.) administration of colchicine on the reappearance of a visually evoked branchial suppression response following axotomy of the goldfish optic nerve were investigated. Fish injected i.o. with 0.1 m g of colchicine within 3 days of optic nerve crush (post crush; PC) recovered vision after some delay relative to recovery of saline-injected lesioned fish, while injection with colchicine between 7 and 14 days PC produced a profound inhibition of visual recovery, in most instances, a complete block for the duration of the study (98 days). Further evidence for a delayed susceptibility to colchicine was reflected in suppression of neurite outgrowth from explanted retinal tissue, in decreased recovery of anterograde transport of labeled proteins and in decreased labeling of retinal ganglion cells (RGCs) from the retrograde transport of a fluorescent dye from the optic tectum. No evidence of residual retinal damage resulting from i.o. colchicine was seen by light microscopy. These results indicate a period of increased vulnerability of the regenerating visual system to colchicine toxicity, beginning 3--5 days PC, and remaining until regenerating optic nerve fibers have reached the tectum. This vulnerable period is correlated with a colchicine-mediated selective decrease of tubulin synthesis. Colchicine injected at the time of crush partially blocks retinal a and b tubulin synthesis, measured two weeks later. There is a concomitant decrease in retinal a and b tubulin mRNAs as established by both RNase protection studies and by <italic>in situ</italic> hybridization of retinal sections, indicating a RGC localization. More profound decreases in tubulin synthesis and mRNA levels are observed in retinas from fish injected during the vulnerable period (7 days PC). By Day 28 PC, fish injected with colchicine at the time of nerve crush showed rates of tubulin synthesis and mRNA levels similar to that seen in saline-injected PC fish. In contrast, fish injected with colchicine during the vulnerable period showed no increases in tubulin mRNA levels up to 44 days PC, a result paralleling the long delay seen in visual recovery. It is concluded that the vulnerable period very likely results from the binding of colchicine to a large pool of tubulin heterodimers present in the RGCs during optic nerve regeneration, which then down-regulates tubulin synthesis and thus inhibits axonal regrowth for long periods of time, perhaps permanently. The temporal consequences of delayed transport of a retrograde signal from the site of crush has significance for both neuroscience and toxicology studies investigating the developing nervous system.