Zinc is a critical regulator of optic nerve regeneration

Abstract

Purpose: Although retinal ganglion cells (RGCs), like most CNS neurons, are normally unable to regenerate injured axons, treatments that synergistically activate RGCs’ intrinsic growth state enable some axons to regenerate through the entire optic nerve and into the brain. However, the number of regenerating axons seen in these studies remains small, implying the existence of additional suppressors of regeneration. Because of studies implicating ionic Zn²⁺ in various neuropathological conditions, we investigated whether it contributes to regulating RGC survival and axon regeneration after optic nerve injury.

Methods: Wild-type C57 mice, Zn²⁺ transporter-3 (ZnT-3) knock-out mice, and phosphatase and tensin homolog (PTEN) conditional knock-out (pten^flx/flx) mice (Jackson Lab, ME) were used in this study. Optic nerve crush (NC) and intraocular injection surgeries were performed on male mice 8 weeks of age. Zn²⁺ autometallography (AMG) and Zinpyr-1 (ZP-1, a specific Zn²⁺ probe) were used to investigate level of ionic Zn²⁺ in the mouse retina at different time-points after NC. Highly selective Zn²⁺ chelators, TPEN or ZX1, were injected intraocularly. Identification of neuronal and synaptic markers of retinal neurons were done by immunostaining in retinal cross-sections. RGC survival in whole-mount retinas and axon regeneration through the optic nerves were quantified at 2 and 12 weeks after NC.

Results: The level of free Zn²⁺ increased in the inner plexiform layer (IPL) of the retina within 1 hour of optic nerve injury and continued to increase up to 24 hours after NC, while levels within RGCs themselves increased more slowly (2~3 days). The vesicular Zn²⁺ transporter protein ZnT-3 was strongly upregulated in the IPL, suggesting that Zn²⁺ becomes sequestered in presynaptic vesicles after injury. Chelating Zn²⁺ using either TPEN or ZX1 or deleting ZnT-3 gene eliminated the Zn²⁺ signal in the IPL and led to enduring survival of RGCs as well as considerable axon regeneration. Combining Zn²⁺ chelation with other pro-regenerative treatments led to extraordinary regeneration, and enabled some RGCs to regenerate damaged axons the full length of the optic nerve in just 2 weeks.

Conclusions: Zn²⁺ is a primary suppressor of RGC death and axon regeneration; Zn²⁺ chelators lead to extensive RGC survival and axon regeneration and may be valuable clinically for various forms of traumatic or neurodegenerative damage to the optic nerve.