Electric Field Stimulation Collaborates with Ciliary Neurotrophic Factor to Direct Retinal Ganglion Cell Axon Regeneration After Optic Nerve Crush Injury

Abstract

Purpose : Neuro-restoration requires strategies that direct both neuronal survival and axon growth, processes that are independently regulated. Our prior work showed that electric field (EF) stimulation with asymmetric charge balanced waveforms directed target-specific regeneration of retinal ganglion cell (RGC) axons after optic nerve crush (ONC) injury but failed to promote RGC survival. A recently published Phase I trial established the safety of a neuroprotective molecule ciliary neurotrophic factor (CNTF). Here, we assessed the potential synergistic effects of combining CNTF and EF stimulation on RGC axon regeneration after ONC injury.

Methods : Adult Long-Evans rats underwent ONC injury with electrode implantation. AAV2-CNTF, CNTF protein, and cAMP were administered intravitreally at the same time as ONC. CNTF protein and cAMP were supplemented three times during the first two weeks after viral transfection. EF stimulation was initiated one week after ONC and continued for 30 days. RGC survival, axon regeneration, electrophysiologic function, and recovery of visual behaviors were assessed in four groups: Untreated sham (n=3), EF alone (n=3), CNTF alone (n=2), and CNTF+EF (n=3).

Results : Untreated rats showed 0.56% ± 0.9% axon regeneration at 250μm past the crush site compared to EF alone resulted in 15.6% ± 3.43%. CNTF alone resulted in 1.5% ± 1.84% regeneration at 250μm past the crush site, while 3.77% ± 1.86% was observed in animals treated with both CNTF and EF. At 1mm past the crush site, untreated rats showed 0.19% ± 0.27% regeneration, EF alone with 3.43% ± 1.46%, CNTF alone with 0.02% ± 0.02%, compared to CNTF+EF with 0.93% ± 0.64%. The normalized ratio of N₉₅ peak amplitude on pattern electroretinogram (PERG) of the crushed to uncrushed eye was 0.30 ± 0.16 in untreated, 0.66 ± 0.14 in EF alone, 0.38 ± 0.007 in CNTF alone, and 0.23 ± 0.18 in CNTF+EF.

Conclusions : Preliminary data showed that combinatorial treatment of EF and CNTF is more effective for axon regeneration than CNTF alone, while EF alone had the best outcome for regeneration. Optical medial opacity due to repeat intravitreal injections within the CNTF groups likely contributed to the findings. Studies in a larger sample size are needed to confirm these findings.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.