Electric Field Application and mTOR Activation Collaborate to Direct Optic Nerve Regeneration After Crush Injury

Abstract

Purpose : Regenerating the optic nerve requires signals that both “fuel” and “steer” retinal ganglion cell (RGC) axon growth to appropriate targets. Although strategies that increase mammalian target of rapamycin (mTOR) activity have been employed to “fuel” RGC axon regeneration, they are not as effective at “steering” regeneration. Based on compelling evidence that electric field (EF) application does both, we investigated whether EFs—in conjunction with mTOR activation—direct more target-specific RGC axon regeneration than with either treatment alone in an optic nerve crush model.

Methods : Adult male Long-Evans rats underwent intravitreal injection with AAV2-rRHEB to increase mTOR activity. Two weeks later, the animals underwent optic nerve crush followed by intra-orbital and intracranial electrode implantation. One week later, biphasic waveforms were applied for 5 hours daily for 30 days. One week before euthanasia, the animals were injected intravitreally with cholera toxin B for anterograde labeling of regenerated axons. Regenerative responses were assessed via histologic, electrophysiologic, behavioral, and pupillary light reflex testing.

Results : Compared to untreated animals (N=5), we observed 33-fold more axon regeneration at 1000 um past the crush site after EF+mTOR treatment (N=2) vs 26-fold with EF (N=5, p=0.03) and 4-fold with mTOR (N=3, p=0.02) (Fig.1; Fig. 2). Local field potential recordings demonstrated greatest partial recovery of visual function in the superior colliculus of EF+mTOR animals with 22.6% of sites responding (N=2), followed by 15.9% in EF (N=6), 1.1% in mTOR (N=3), and 1.3% in untreated animals (N=5) (EF vs untreated, p ≤ 0.001). Each animal tested for pupillary light reflex in the EF+mTOR (N=6) and EF (N=10) groups consistently demonstrated pupillary constriction whereas only 33.3% in the untreated (N=6) and mTOR (N=3) groups did.

Conclusions : The results suggest that treatment with EF exerts synergistic effects with mTOR in promoting RGC survival and directing target-specific long distance axon growth, with consequent partial recovery of visual function in rats.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Figure 1. Varying levels of regenerated RGC axons (white) observed after different treatments, with EF+mTOR demonstrating greatest efficacy. Asterisk denotes crush site and scale bar is 250um.

 

Figure 2. Long distance axon regeneration seen in the optic chiasm (arrow) after treatment with EF+mTOR.