Abstract
Purpose: :
Injured CNS including optic nerve has been known of incapable to regenerate the injured axons in adult mammal. In addition to the known astrocyte- and myelin-related growth inhibitors along CNS pathway, the infiltration of leucocytes may play a role to block optic nerve regeneration. It has been shown that upregulation of CCL2 would regulate infiltration of leucocytes into the lesion area. To test this hypothesis, we performed optic nerve crush in adult C57BL/6J and CCL2 knockout mice and compare the axonal regrowth after injury.
Methods: :
Optic nerve crush was performed in adult CCL2 knockout and C57BL/6J (background-matched control) mice. On day 11 post-injury, 2 µl of an anterograde axon tracer, cholera toxin B subunit (CTB), was injected into the vitreous of the operated eye. On day 14 post-injury, mice were sacrificed and optic nerves were collected and processed for cryosections. To determine axonal regrowth, the sections were incubated with goat anti-CTB antibody, followed by secondary antibody of biotinylated anti-goat IgG, and were visualized by Alexa Fluor 546. To determine retinal ganglion cell survival, retinal sections were incubated with primary antibody against beta-III-tubulin and 4', 6-diamidino-2-phenylindole (DAPI). Number of CTB-positive axons that regenerated past the lesion site was counted at steps of 250 µm away from the crushed site in optic nerve sections. Numbers of beta-III-tubulin- and DAPI-positive cells in the ganglion cell layer were counted in 5 random and non-overlapping areas in each retinal section under 400x magnifications.
Results: :
Little axonal regeneration was observed in wild-type mice. However, absence of CCL2 significantly improved axonal regrowth after optic nerve injury in CCL2 knockout mice as compared to wild-type mice, and the number of regenerating axons counted from CCL2 knockout mice was ten times more than was observed in wild-type mice.
Conclusions: :
Absence of CCL2 provides a permissive environment for axonal sprouting, likely through the suppression of injury-induced activation of microglia and macrophages.
Keywords: optic nerve • regeneration • inflammation